Combination tumor treatment with an integrin-binding-fc fusion protein and immune stimulator

ABSTRACT

The present invention provides a method of treating cancer with an integrin-binding-Fc fusion protein alone or in combination with IL-2 and/or an immune stimulant (i.e., an immune checkpoint stimulator), and/or an immune checkpoint inhibitor. The invention also provides composition for use in such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of Ser. No. 15/867,655 filedJan. 10, 2018, now U.S. Pat. No. 10,603,358, which claims priority toU.S. Provisional Application No. 62/444,660, filed Jan. 10, 2017; U.S.Provisional Application No. 62/466,298 filed Mar. 2, 2017; U.S.Provisional Application No. 62/500,203 filed May 2, 2017; U.S.Provisional Application No. 62/523,191 filed Jun. 21, 2017; U.S.Provisional Application No. 62/523,200 filed Jun. 21, 2017; U.S.Provisional Application No. 62/573,079 filed Oct. 16, 2017; and U.S.Provisional Application No. 62/580,783 filed Nov. 2, 2017, thedisclosures of which are hereby incorporated by reference in theirentireties for all purposes.

BACKGROUND OF THE INVENTION

Interleukin-2 (IL-2) is a pleiotropic cytokine that activates andinduces the proliferation of T cells and NK cells. Although IL-2 is anFDA approved therapy, systemic IL-2 treatment has significant toxicityand the response rate of patients is less than 25%. Combining IL-2and/or extended half-life IL-2 and an antibody against a tumor-specificantigen to invoke the adaptive and innate arms of the immune systemshows promising results for treatment. However, antibody-based therapiesoften suffer from the fact that many tumors lack known tumor-associatedantigens.

Integrins are a family of extracellular matrix adhesion receptors thatregulate a diverse array of cellular functions crucial to theinitiation, progression and metastasis of solid tumors. The importanceof integrins in tumor progression has made them an appealing target forcancer therapy and allows for the treatment of a variety of cancertypes. The integrins present on cancerous cells include α_(v)β₃,α_(v)β₅, and α₅β₁. A variety of therapeutics have been developed totarget individual integrins associated with cancer, includingantibodies, linear peptides, cyclic peptides, and peptidomimetics.However, none have utilized small, structured peptide scaffolds ortargeted more than two integrins simultaneously. Additionally, currentintegrin targeting drugs are given as a monotherapy. Novel monotherapiesas well as combination therapies are needed to more effectively combatvarious cancers.

The present invention meets this need and provides novel monotherapiesand combination therapies for use in cancer treatment.

BRIEF SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatadministration of an integrin-binding polypeptide-Fc fusions describedherein can be useful in the treatment of cancer.

The present invention provides methods for treating cancer in a subjectcomprising administering to the subject an effective amount of anintegrin-binding polypeptide-Fc fusion wherein said integrin-bindingpolypeptide-Fc fusion is administered in a therapeutically effectiveamount, wherein said integrin-binding polypeptide comprises a sequenceselected from the group consisting of SEQ ID NO:130(GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG) and SEQ ID NO:131(GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG) and wherein said integrin-bindingpolypeptide is conjugated to an Fc domain.

In some embodiments, the Fc domain is selected from the group consistingof IgG1, IgG2, IgG3, and IgG4.

In some embodiments, the said Fc domain is a human Fc domain.

In some embodiments, the integrin-binding polypeptide is conjugateddirectly to said Fc domain.

In some embodiments, the integrin-binding polypeptide is conjugated tosaid Fc domain through a linker polypeptide.

In some embodiments, the linker polypeptide is selected from the groupconsisting of GGGGS (SEQ ID NO:136) and GGGGSGGGGSGGGGS (SEQ ID NO:137).

In some embodiments, the method further comprises administering animmune checkpoint inhibitor. In some embodiments, the method furthercomprises administering an immune checkpoint stimulator.

In some embodiments, the immune checkpoint inhibitor is a PD-1inhibitor.

In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.

In some embodiments, the integrin-binding polypeptide-Fc fusioncomprises an integrin-binding polypeptide sequence in the presence orabsence of a linker, wherein said sequence is selected from the groupconsisting of GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.

In some embodiments, the method further comprises administering aninterleukin-2 (IL-2).

In some embodiments, the IL-2 is Proleukin.

In some embodiments, the IL-2 is administered before, after orsimultaneously with administration of said integrin-bindingpolypeptide-Fc fusion.

In some embodiments, the IL-2 is administered after administration ofsaid integrin-binding polypeptide-Fc fusion.

In some embodiments, the IL-2 is administered at a 12 MIU/m2 or lowerdaily dose.

In some embodiments, the IL-2 is administered subcutaneously.

In some embodiments, the method further comprises administering eitherIL-2 or an immune checkpoint inhibitor. In some embodiments, the methodfurther comprises administering either IL-2 or an immune checkpointstimulator.

In some embodiments, the method further comprises administering IL-2 andan immune checkpoint inhibitor. In some embodiments, the method furthercomprises administering IL-2 and an immune checkpoint stimulator.

In some embodiments, the immune checkpoint inhibitor is selected fromthe group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody,and an anti-CTLA-4 antibody. In some embodiments, the immune checkpointstimulator is selected from the group consisting of an anti-4-1BB/CD137antibody, an anti-IFNα antibody, an anti-GITR antibody, and an OX40antibody. In some embodiments, the immune checkpoint inhibitor is ananti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitoris an anti-PD-L1 antibody. In some embodiments, the immune checkpointinhibitor is an anti-CTLA-4 antibody. In some embodiments, the immunecheckpoint stimulator is an anti-4-1BB/CD137 antibody.

In some embodiments, the said integrin-binding polypeptide-Fc fusionbinds to at least two integrins.

In some embodiments, the integrin-binding polypeptide-Fc fusion binds toat least three integrins.

In some embodiments, the integrin-binding polypeptide-Fc fusion binds toat least two integrins selected from the group consisting of α_(v)β₁,α_(v)β₃, α_(v)β₅, α_(v)β₆, and α₅β₁.

In some embodiments, the further administration of IL-2 or an immunecheckpoint inhibitor induces tumor infiltration of CD8+ T-cells ascompared to non-administration. In some embodiments, the furtheradministration of IL-2 or an immune checkpoint stimulator induces tumorinfiltration of CD8+ T-cells as compared to non-administration. In someembodiments, the further administration of IL-2 or an anti-PD-1 antibodyinduces tumor infiltration of CD8+ T-cells as compared tonon-administration.

In some embodiments, the further administration of IL-2 or an immunecheckpoint inhibitor induces a decrease in myeloid-derived suppressorcells (MDSC) as compared to non-administration. In some embodiments, thefurther administration of IL-2 or an immune checkpoint stimulatorinduces a decrease in myeloid-derived suppressor cells (MDSC) ascompared to non-administration. In some embodiments, the furtheradministration of IL-2 or an anti-PD-1 antibody induces a decrease inmyeloid-derived suppressor cells (MDSC) as compared tonon-administration.

In some embodiments, the further administration of both IL-2 and animmune checkpoint inhibitor induces increased tumor infiltration of CD8+T-cells as compared to administration of an IL-2 and/or an immunecheckpoint inhibitor individually. In some embodiments, the furtheradministration of both IL-2 and an immune checkpoint stimulator inducesincreased tumor infiltration of CD8+ T-cells as compared toadministration of an IL-2 and/or an immune checkpoint inhibitorindividually. In some embodiments, the further administration of bothIL-2 and an anti-PD-1 antibody induces increased tumor infiltration ofCD8+ T-cells as compared to administration of an IL-2 and/or ananti-PD-1 antibody individually.

In some embodiments, the further administration of both IL-2 and animmune checkpoint inhibitor induces a greater decrease inmyeloid-derived suppressor cells (MDSC) cells as compared toadministration of an IL-2 and/or an immune checkpoint inhibitorindividually. In some embodiments, the further administration of bothIL-2 and an immune checkpoint stimulator induces a greater decrease inmyeloid-derived suppressor cells (MDSC) cells as compared toadministration of an IL-2 and/or an immune checkpoint stimulatorindividually. In some embodiments, the further administration of bothIL-2 and an anti-PD-1 antibody induces a greater decrease inmyeloid-derived suppressor cells (MDSC) cells as compared toadministration of an IL-2 and/or an anti-PD-1 antibody individually.

The present invention also provides polypeptides comprising anintegrin-binding polypeptide sequence in the presence or absence of alinker, wherein said sequence is selected from the group consisting ofGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.

The present invention also provides compositions comprising anintegrin-binding polypeptide sequence in the presence or absence of alinker, wherein said sequence is selected from the group consisting ofGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.

The present invention also provides pharmaceutical compositionscomprising an integrin-binding polypeptide sequence in the presence orabsence of a linker, wherein said sequence is selected from the groupconsisting of GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.

The present invention also provides nucleic acids encoding anintegrin-binding polypeptide-Fc fusion as described herein.

The present invention also provides expression vectors comprising anucleic acid encoding an integrin-binding polypeptide-Fc fusion asdescribed herein.

The present invention also provides host cells comprising the expressionvector of claim 31. The present invention further provides for a methodof making an integrin-binding polypeptide-Fc fusion as described hereincomprising

-   -   a) culturing the host cell of claim 32 under conditions wherein        said integrin-binding polypeptide-Fc fusion is expressed; and    -   b) recovering said integrin-binding polypeptide-Fc fusion.    -   The present invention provides methods for activating the immune        system in order to treat cancer in a subject comprising        administering to the subject an effective amount of an        integrin-binding polypeptide-Fc fusion, wherein said        integrin-binding polypeptide-Fc fusion is administered in a        therapeutically effective amount, wherein said integrin-binding        polypeptide comprises a sequence selected from the group        consisting of SEQ ID NO:130 (GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG)        and SEQ ID NO:131 (GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG), and        wherein said integrin-binding polypeptide is conjugated to an Fc        domain.

In some embodiments, the Fc domain is selected from the group consistingof IgG1, IgG2, IgG3, and IgG4.

In some embodiments, the Fc domain is a human Fc domain.

In some embodiments, the integrin-binding polypeptide is conjugateddirectly to said Fc domain.

In some embodiments, the integrin-binding polypeptide is conjugated tosaid Fc domain through a linker polypeptide.

In some embodiments, the linker polypeptide is selected from the groupconsisting of GGGGS (SEQ ID NO:136) and GGGGSGGGGSGGGGS (SEQ ID NO:137).

BRIEF DESCRIPTION OF THE DRAWINGS

-   -   These and other features, aspects, and advantages of the present        invention will become better understood with regard to the        following description, and accompanying drawings.

FIG. 1A-FIG. 1B. provides examples of IgG1, IgG2, IgG3, and IgG4sequences.

FIG. 2. NOD201, a “pseudo-mAb” created by fusing an engineered cystineknot (knottin) peptide to an Fc domain. This construct targets innateeffector functions (ADCC and CDC) against α_(v)β₁, α_(v)β₃, α_(v)β₅,α_(v)β₆, and α₅β₁ integrin.

FIG. 3A-FIG. 3B. NOD201M has weak efficacy as a monotherapy. Left, Tumorvolume curves Right, Kaplan-Meier curves. NOD201M was administered IV atdoses of 250 μg, 500 μg, or 1000 μg on days 1, 7, 13, 19 (i.e., once perweek) after inoculated tumors reached an average size of 60-180 mm³.Vehicle: phosphate buffered saline. MC38 colon tumor model.

FIG. 4A-FIG. 4B. NOD201M potentiates low dose IL-2 (4 Proleukin). Left,Tumor volume curves. Right, Kaplan-Meier curves. NOD201M wasadministered IV at doses of 500 μg or 1000 μg on days 1, 7, 13, 19 afterinoculated tumors reached an average size of 60-180 mm³. IL-2 (4Proleukin) was administered subcutaneously on days 2-4, 8-10, 14-16,20-22 (days +1, +2, +3). Vehicle: phosphate buffered saline. MC38 colontumor model.

FIG. 5A-FIG. 5B. NOD201M potentiates anti-PD-1. Left, Tumor volumecurves. Right, Kaplan-Meier curves. NOD201M was administered IV at dosesof 250 μg, 500 μg, or 1000 μg on days 1, 7, 13, 19 after inoculatedtumors reached an average size of 60-180 mm³. Anti-PD-1 (200 μg; cloneRMP1-14) was administered IV on days 1, 7, 13, 19. Vehicle: phosphatebuffered saline. MC38 colon tumor model.

FIG. 6A-FIG. 6B. Tumor cell infiltrates measured following NOD201Mcombination therapy. Left, NOD201M combination therapy results in asignificant increase of CD8+ T cells in the tumor following combinationtreatment with anti-PD, low dose IL-2, or both. Right, Myeloid-derivedsuppressor cells measured in the tumor following treatment. NOD201M,anti-PD-1, and low dose IL-2 was administered as described in FIG. 11.Tumors were stained and analyzed for cell surface markers by flowcytometry at day 9; 24 hours post Proleukin dose. Measurements weretaken 2 days following 2nd dose cycle. MC38 tumor model.

FIG. 7A-FIG. 7J. NOD201M efficacy in the B16F10 melanoma model. Tumorvolume curves for NOD201M+/−IL-2+/−anti-PD-1 combination therapy. A)NOD201M. B) Anti-PD-1 antibody. C) IL-2. D) NOD201M+anti-PD-1 antibody.E) NOD201M+IL-2. F) NOD201M+anti-PD-1 antibody+IL-2. G)/J Graphicalrepresentation of the data. In this experiment, the study start date wasthe day of tumor implant (Day 1) and not the first day of dosing ofestablished tumors as in MC38 studies. NOD201M was administered IV atdoses of 500 μg or 1000 μg (as indicated) on days 4, 10, 16, 22. IL-2 (4μg; Proleukin) was administered subcutaneously on days 5-7, 11-13,17-19, 23-25. Anti-PD-1 (200 μg; clone RMP1-14) was administered IV ondays 4, 10, 16, 22. B16F10 tumor model.

FIG. 8A-FIG. 8C. Analytical characterization of NOD201 produced from atransient HEK expression system. SDS-PAGE of expressed and purifiedNOD201 showed bands of the expected molecular weight; reduced andnon-reduced samples were analyzed. Size exclusion chromatography ofpurified NOD201 following mAbSelect SuRe resin. RP-HPLC of purifiedNOD201 formulated in PBS, pH 7.4 using a C8 column. Thermal stability:68° C. in PBS, pH 7.4 as measured by DSF.

FIG. 9A-FIG. 9D. NOD201 binds with high affinity to cells expressinghuman and monkey integrins. NOD201X contains a scrambled binding epitopeand serves as a negative control. U87MG (human tumor cells); CV-1(monkey); RF/6A (monkey). Binding was measured using an antibody againstthe NOD201 or NOD201X Fc domain using flow cytometry. Binding of NOD201Mto U87MG cells was also measured for comparison and shows similarbinding affinity as NOD201.

FIG. 10A-FIG. 10C. NOD201 binds with high affinity to cells expressingrat and rabbit integrins. NOD201X contains a scrambled binding epitopeand serves as a negative control. Rat2 (rat fibroblasts); SIRC (rabbitcorneal cells). Binding was measured using an antibody against theNOD201 or NOD201X Fc domain using flow cytometry.

FIG. 11A-11D. Tumor volume (left) and Kaplan-Meier curves (right) forNOD201M+IL-2+anti-PD-1 combination therapy. MC38 colon tumor model.NOD201M was administered IV at doses of 250 μg, 500 μg, or 1000 μg ondays 1, 7, 13, 19 after inoculated tumors reached an average size of60-180 mm³. IL-2 (4 Proleukin) was administered subcutaneously on days2-4, 8-10, 14-16, 20-22. Anti-PD-1 (200 μg; clone RMP1-14) wasadministered IV on days 1, 7, 13, 19.

FIG. 12A-FIG. 12B. Addition of IL-2 to NOD201M+anti-PD-1 combinationtherapy does not improve 30-day survival. MC38 colon tumor model.

FIG. 13. Study design to determine the efficacy of NOD201M alone and incombination with high dose or low dose Proleukin, and anti-PD-1 in theMC38-NODU syngeneic colon model using female C57BL/6 mice. Day indicatesdosing administered after inoculated tumors reached an average size of60-180 mm³.

FIG. 14A-FIG. 14B. NOD201 binds to multiple murine and human RGD-bindingintegrin heterodimers with high affinity.

FIG. 15. Individual times to end-point for mice in the study designoutlined in FIG. 13. NOD201M, Proleukin, or anti-PD-1 were administeredas indicated.

FIG. 16A-FIG. 16B. Median tumor growth and Kaplan-Meier plots for micein the study design outlined in FIG. 13. NOD201M, Proleukin, oranti-PD-1 were administered as indicated.

FIG. 17. Mean tumor volume curves for mice in the study design outlinedin FIG. 13. NOD201M, Proleukin, or anti-PD-1 were administered asindicated.

FIG. 18A-FIG. 18D. Individual tumor volume growth curves for mice in thestudy design outlined in FIG. 13.

FIG. 19A-FIG. 19D. Individual tumor volume growth curves for mice in thestudy design outlined in FIG. 13.

FIG. 20A-FIG. 20C. Individual tumor volume growth curves for mice in thestudy design outlined in FIG. 13.

FIG. 21. Percent group mean body weight for mice in the study designoutlined in FIG. 13.

FIG. 22A-FIG. 22B. NOD201 dosing rationale. >25 mg/kg in mice and >10mg/kg in man required to overcome 1) rapid systemic clearance (˜60 kDa),2) rapid endocytic clearance (˜1.5 hr half-life), 3) TMDD (PBMC) Annalsof oncology 2013; 24(2):329-36. Model calculations as per J Theor. Biol.314:57-68 (2012).

FIG. 23A-FIG. 231. Tumor volume curves for various treatments ortreatment combinations. A) Saline (control). B) NOD201M C)NOD201M+anti-PD-1 antibody, D) NOD201M+anti-PD-L1 antibody, E)NOD201M+anti-CTLA-4 antibody, F) NOD201M+anti-LAG-3 antibody, G)NOD201M+anti-TIM-3 antibody, H) NOD201M+anti-TIGIT antibody, and I)NOD201M+anti-4-1BB/CD137 antibody. Increased effects on reducing tumorvolume over the effects observed with NOD201M alone were observed forNOD201M+anti-PD-1 antibody, NOD201M+anti-PD-L1 antibody,NOD201M+anti-CTLA-4 antibody, and NOD201M+anti-4-1BB/CD137 antibody.Increased effects on reducing tumor volume over the effects observedwith NOD201M alone were not observed for NOD201M+anti-LAG-3 antibody,NOD201M+anti-TIM-3 antibody, and NOD201M+anti-TIGIT antibody.

FIG. 24. Survival curves for various treatment combinations ofNOD201M+/−various checkpoint inhibitors (an anti-PD-L1 an antibody, ananti-4-1BB/CD137 antibody, an anti-CTLA-4 antibody, an anti-PD-1antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or ananti-TIGIT antibody). Increased survival effects were observed withcombinations between NOD201M+anti-CTLA-4 antibody, NOD201M+anti-PD-L1antibody, NOD201M+anti-4-1BB/CD137 antibody, and NOD201M+anti-PD-1antibody as compared to survival with NOD201M alone. Increased survivaleffects were not observed with NOD201M+anti-LAG-3 antibody,NOD201M+anti-TIM-3 antibody, or NOD201M+anti-TIGIT antibody.

FIG. 25A-FIG. 25M. Tumor cell infiltrates resulting from NOD201Mcombination therapy. Measured 2 days following 2nd dose cycle (day 9).A) CD8, B) CD4 (n.s.), C) Treg (n.s.), D) Macrophages, E) M1macrophages, F) M2 macrophages, G) NK (n.s.), H) Dendritic cells (n.s.),I) MHCII−, J) MHCII+ (n.s.), K) MDSC (n.s.), L) gMDSC (n.s.), and M)mMDSC (n.s.). A) CD8, D) E) M1 macrophages for the combo treatment, andI) M2 macrophages for αPD-1 exhibited statistical significance. CD8T-cells and macrophages are involved in the therapeutic effect withNOD201M and αPD-1. However, single agent NOD201M or αPD-1 did notexhibit a statistically significant change in the number of CD8 T-cellsor macrophages in the tumor. Cells that did not change significantlyupon treatment with single agent or combo were CD4 T-cells, Tregs, NKcells, MDSC, gMDSC, mMDSC, dendritic cells, and MHCII+. M2 macrophages:these tolerizing or immunosuppressive macrophages were increased withαPD-1 treatment alone. The combo arm was lowered in M2 back down tosaline treated levels. M1 macrophages: these highly stimulated tumorkilling macrophage type were increased with the combo as compared to nodifference with single agents. MHCII− (likely dendritic cells) do changewith the combo treatment but not single agent treatment.

FIG. 26A-FIG. 26B. Tumor cell infiltrates resulting from NOD201Mcombination therapy with αPD-1 and LD IL-2. A) CD8. B) MDSC. Measured 2days following 2nd dose cycle. MC38 colon tumor model.

FIG. 27A-FIG. 27B. NOD201 effectively combines with αPD-1, αPD-L1,αCTLA-4, and α4-1BB/CD137. Corresponding monotherapies non-effective.+αTIM3, +αTIGIT, +αLAG3: no incremental effect.

FIG. 28. Combination therapy invokes innate and adaptive immune system.NOD201-D265A: a point mutation in the Fc domain (D265A) disrupts FcRybinding and therapeutic efficacy with the anti-PD1 combo therapy,showing that effector functions are required. Fc effector functionsdrives cross priming of T cell response—vaccinal effect. This utilizes:Macrophages, CD8+ T cells, and CD8+ dendritic cells. Fc effectorfunctions create inflammatory tumor microenvironment—increasedintratumoral chemokines.

FIG. 29A-FIG. 29D. Human colon tumor 1 tissue staining is shown. NOD201,α5 integrin, NOD201X (negative control), and αv integrin. Tissuereactivity studies underway (integrin profiling and NOD201 staining onhealthy tissue in progress).

FIG. 30A-FIG. 30D. Human colon tumor 2 tissue staining is shown. NOD201,α5 integrin, NOD201X (negative control), and αv integrin.

FIG. 31A-FIG. 31D. Human prostate tumor 1 tissue staining is shown.NOD201, α5 integrin, NOD201X (negative control), and αv integrin.

FIG. 32A-FIG. 32D. Human pancreatic tumor 1 tissue staining is shown.NOD201, α5 integrin, NOD201X (negative control), and αv integrin.

FIG. 33A-FIG. 33D. Human glioblastoma tumor 1 tissue staining is shown.NOD201, α5 integrin, NOD201X (negative control), and αv integrin.

FIG. 34A-FIG. 34C. NOD201+αPD1 combination therapy can recruit T cellsinto the tumor and reduce MDSCs. NOD201+αPD1 in the MC38 colon tumormodel analyzed at different time points post treatment.

FIG. 35A-FIG. 35B. Patient “responder” gene signature correlates withmouse tumors that are the best responders. Paired gene analysis inpatient non-responders (NR) and responders (R) treated with α-PD1. Tumorarray analyses were performed (integrin profiling and NOD201 staining ontumor panels, see also FIGS. 29-33). A) Paired gene analysis identifieda panel of genes that were upregulated in patients that responded totreatment with anti-PD1 (R) compared to non-responders (NR). Data takenfrom Chen et al. Cancer Discovery 2016, 6:82. B) Similar genes wereanalyzed by RNAseq from MC38 syngeneic tumors treated with salinecontrol, anti-PD1 alone, NOD201 alone, or the combination of anti-PD1and NOD201, as described in Example 7. Change in tumor volume wasmeasured over day 5 to day 9 with largest tumors termed non-respondersand smallest tumors termed responders. Data was grouped according totumor volume and plotted as a heat map indicating bulk gene expressionin the tumor. Mouse tumors that were responders exhibited immune genesignatures that are similar to those identified in the Chen 2016 studyas patient responder.

FIG. 36. Immune response gene signature correlates with tumors that arethe best responders. T-cell activation genes are upregulated withresponders, downregulated with non-responders. Chen checkpoint signaturecorrelates with tumor volume change. Gene set scores, plotted againstthe tumor volume changes from Day 5 to Day 9, according to treatmentgroup.

FIG. 37A-FIG. 37B. Chen et al., immune response gene signatures areenriched only when αPD1 was added to NOD201.

FIG. 38. RNAseq analysis data which shows that genes involved in T-cellactivation are upregulated in response to treatment with NOD201+αPD1,while some T-cell activation genes are downregulated in response totreatment with αPD1 alone. T-cell activation genes are upregulated withresponders, downregulated with non-responders. Clustered according togenes.

FIG. 39. RNAseq analysis data which shows that genes involved in T-cellactivation are upregulated in response to treatment with NOD201+αPD1,while some T-cell activation genes are downregulated in response totreatment with αPD1 alone. (Data ordered by volume.) T cell activationgenes are upregulated with responders, downregulated withnon-responders. Samples were grouped according to change in tumor volumeover days 5 to day 9, with responders corresponding to tumors that hadthe smallest volume change over time.

FIG. 40A-FIG. 40E. 2.5F knottin peptide fused to different chains andtermini of an antibody.

FIG. 41A-FIG. 41H. 2.5F knottin peptide fused to different chains andtermini of an anti-CTLA-4 antibody.

FIG. 42A-FIG. 42B. Survival curves for various treatment combinations ofNOD201M+/−various checkpoint inhibitors (an anti-PD-L1 antibody, ananti-4-1BB/CD137 antibody, an anti-CTLA-4 antibody, an anti-PD-1antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or ananti-TIGIT antibody) and an IFN-α. Increased survival effects wereobserved with combinations between NOD201M+anti-CTLA-4 antibody,NOD201M+anti-PD-L1 antibody, NOD201M+anti-4-1BB/CD137 antibody,NOD201M+anti-PD-1 antibody, and NOD201M+an IFN-α as compared to survivalwith NOD201M alone. Increased survival effects were not observed withNOD201M+anti-LAG-3 antibody, NOD201M+anti-TIM-3 antibody, orNOD201M+anti-TIGIT antibody.

FIG. 43A-FIG. 43H. α5 and αv integrin: patient tumor staining profile. %patient tumor samples that stained positive for α5 or αv integrin. n=20for breast cancer, head and neck, NSCLC, pancreatic cancer, and GBM.n=16 for melanoma, n=24 for colon cancer. No α5 staining shown forbreast cancer.

FIG. 44A-FIG. 44G. Aggregated patient data for tumor and fibrovascularstaining of α5 and αv integrins, aggregate from FIG. 43 data. A) Breastcancer. B) Head and neck cancer. C) Melanoma. D) NSCLC. E) Colon cancer.F) Pancreatic cancer. G) GBM.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Interleukin-2 (IL-2) is a pleiotropic cytokine that activates andinduces the proliferation of T cells and NK cells. Although IL-2 is anFDA approved therapy, systemic IL-2 treatment has significant toxicityand the response rate of patients is less than 25%. Combining extendedhalf-life IL-2 and an antibody against a tumor-specific antigen showspromising results for treatment. However, antibody-based therapies oftensuffer from the fact that many tumors lack known tumor-associatedantigens.

Integrins are a family of extracellular matrix adhesion receptors thatregulate a diverse array of cellular functions crucial to theinitiation, progression and metastasis of solid tumors. The importanceof integrins in tumor progression has made them an appealing target forcancer therapy and allows for the treatment of a variety of cancertypes. The integrins present on cancerous cells include α_(v)β₃,α_(v)β₅, and α₅β₁. A variety of therapeutics have been developed totarget individual integrins associated with cancer, includingantibodies, linear peptides, cyclic peptides, and peptidomimetics.However, none have utilized small, structured peptide scaffolds ortargeted more than two integrins simultaneously. Additionally, currentintegrin targeting drugs are given as a monotherapy. Novel combinationtherapies are needed to more effectively combat various cancers.

II. Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant specification shall control.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid. Amino acids can be referred to herein byeither their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, can be referred to by their commonlyaccepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, the presentlarger “peptide insertions,” can be made, e.g. insertion of about threeto about five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992;Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine andleucine, modifications at the second base can also be conservative. Theterm nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene. Polynucleotides used herein can be composed of anypolyribonucleotide or polydeoxribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, “interleukin (IL)-2,” refers to a pleiotropic cytokinethat activates and induces proliferation of T cells and natural killer(NK) cells. IL-2 signals by binding its receptor, IL-2R, which iscomprised of alpha, beta, and gamma subunits. IL-2 signaling stimulatesproliferation of antigen-activated T cells.

As used herein, the term “PK” is an acronym for “pharmacokinetic” andencompasses properties of a compound including, by way of example,absorption, distribution, metabolism, and elimination by a subject. Asused herein, an “extended-PK group” refers to a protein, peptide, ormoiety that increases the circulation half-life of a biologically activemolecule when fused to or administered together with the biologicallyactive molecule. Examples of an extended-PK group include PEG, humanserum albumin (HSA) binders (as disclosed in U.S. Publication Nos.2005/0287153 and 2007/0003549, PCT Publication Nos. WO 2009/083804 andWO 2009/133208, and SABA molecules as described in US Publication No.2012/094909), human serum albumin, Fc or Fc fragments and variantsthereof, and sugars (e.g., sialic acid). Other exemplary extended-PKgroups are disclosed in Kontermann et al., Current Opinion inBiotechnology 2011; 22:868-876, which is herein incorporated byreference in its entirety. As used herein, an “extended-PK IL-2” refersto an IL-2 moiety in combination with an extended-PK group. In oneembodiment, the extended-PK IL-2 is a fusion protein in which an IL-2moiety is linked or fused to an extended-PK group. An exemplary fusionprotein is an HSA/IL-2 fusion in which one or more IL-2 moieties arelinked to HSA.

The term “extended-PK IL-2” is also intended to encompass IL-2 mutantswith mutations in one or more amino acid residues that enhance theaffinity of IL-2 for one or more of its receptors, for example, CD25. Inone embodiment, the IL-2 moiety of extended-PK IL-2 is wild-type IL-2.In another embodiment, the IL-2 moiety is a mutant IL-2 which exhibitsgreater affinity for CD25 than wild-type IL-2. When a particular type ofextended-PK group is indicated, such as HSA-IL-2, it should beunderstood that this encompasses both HSA or MSA fused to a wild-typeIL-2 moiety or HSA or MSA fused to a mutant IL-2 moiety.

In certain aspects, the extended-PK IL-2 or knottin-Fc described canemploy one or more “linker domains,” such as polypeptide linkers. Asused herein, the term “linker” or “linker domain” refers to a sequencewhich connects two or more domains (e.g., the PK moiety and IL-2) in alinear sequence. As used herein, the term “polypeptide linker” refers toa peptide or polypeptide sequence (e.g., a synthetic peptide orpolypeptide sequence) which connects two or more domains in a linearamino acid sequence of a polypeptide chain. For example, polypeptidelinkers may be used to connect an IL-2 moiety or an integrin-bindingpolypeptide to an Fc domain or other PK-extender such as HSA. In someembodiments, such polypeptide linkers can provide flexibility to thepolypeptide molecule. Exemplary linkers include Gly-Ser linkers, such asbut not limited to [Gly4Ser], comprising 4 glycines followed by 1 serineand [Gly4Ser3], comprising 4 glycines followed by 3 serines.

As used herein, the terms “linked,” “fused”, or “fusion” are usedinterchangeably. These terms refer to the joining together of two ormore elements or components or domains, by whatever means includingchemical conjugation or recombinant means. Methods of chemicalconjugation (e.g., using heterobifunctional crosslinking agents) areknown in the art.

The term “integrin” means a transmembrane heterodimeric proteinimportant for cell adhesion. Integrins comprise an α and β subunit.These proteins bind to extracellular matrix components (e.g.,fibronectin, collagen, laminin, etc.) and respond by inducing signalingcascades. Integrins bind to extracellular matrix components byrecognition of an Arg-Gly-Asp (RGD) motif. Certain integrins are foundon the surface of tumor cells and therefore make promising therapeutictargets. In certain embodiments, the integrins being targeted areα_(v)β₃, α_(v)β5, and α5β1, individually or in combination.

The term “integrin-binding polypeptide” refers to a polypeptide whichincludes an integrin-binding domain or loop within a knottin polypeptidescaffold. The integrin binding domain or loop includes at least one RGDpeptide. In certain embodiments, the RGD peptide is recognized byα_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, and α₅β₁ integrins. In certainembodiments the RGD peptide binds to a combination of α_(v)β₁, α_(v)β₃,α_(v)β₅, α_(v)β₆, and α₅β₁ integrins. These specific integrins are foundon tumor cells and their vasculature and are therefore the targets ofinterest.

Integrins are a family of extracellular matrix adhesion proteins thatnoncovalently associate into α and β heterodimers with distinct cellularand adhesive specificities (Hynes, 1992; Luscinskas and Lawler, 1994).Cell adhesion, mediated though integrin-protein interactions, isresponsible for cell motility, survival, and differentiation. Each α andβ subunit of the integrin receptor contributes to ligand binding andspecificity.

Protein binding to many different cell surface integrins can be mediatedthrough the short peptide motif Arg-Gly-Asp (RGD) (Pierschbacher andRuoslahti, 1984). These peptides have dual functions: They promote celladhesion when immobilized onto a surface, and they inhibit cell adhesionwhen presented to cells in solution. Adhesion proteins that contain theRGD sequence include: fibronectin, vitronectin, osteopontin, fibrinogen,von Willebrand factor, thrombospondin, laminin, entactin, tenascin, andbone sialoprotein (Ruoslahti, 1996). The RGD sequence displaysspecificity to about half of the 20 known integrins including the α₅β₁,α₈β₁, α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, and α_(v)β₃integrins, and, to a lesser extent, the α₂β₁, α₃β₁, α₄β₁, and α₇β₁integrins (Ruoslahti, 1996). In particular, the α_(v)β₃ integrin iscapable of binding to a large variety of RGD containing proteinsincluding fibronectin, fibrinogen, vitronectin, osteopontin, vonWillebrand factor, and thrombospondin (Ruoslahti, 1996; Haubner et al.,1997), while the α₅β₁ integrin is more specific and has only been shownto bind to fibronectin (D'Souza et al., 1991).

The linear peptide sequence RGD has a much lower affinity for integrinsthan the proteins from which it is derived (Hautanen et al., 1989). Thisdue to conformational specificity afforded by folded protein domains notpresent in linear peptides. Increased functional integrin activity hasresulted from preparation of cyclic RGD motifs, alteration of theresidues flanking the RGD sequence, and synthesis of small moleculemimetics (reviewed in (Ruoslahti, 1996; Haubner et al., 1997)).

The term “loop domain” refers to an amino acid subsequence within apeptide chain that has no ordered secondary structure, and residesgenerally on the surface of the peptide. The term “loop” is understoodin the art as referring to secondary structures that are not ordered asin the form of an alpha helix, beta sheet, etc.

The term “integrin-binding loop” refers to a primary sequence of about9-13 amino acids which is typically created ab initio throughexperimental methods such as directed molecular evolution to bind tointegrins. In certain embodiments, the integrin-binding loop includes anRGD peptide sequence, or the like, placed between amino acids which areparticular to the scaffold and the binding specificity desired. TheRGD-containing peptide or similar peptide (such as RYD, etc.) isgenerally not simply taken from a natural binding sequence of a knownprotein. The integrin-binding loop is preferably inserted within aknottin polypeptide scaffold between cysteine residues, and the lengthof the loop adjusted for optimal integrin-binding depending on thethree-dimensional spacing between cysteine residues. For example, if theflanking cysteine residues in the knottin scaffold are linked to eachother, the optimal loop may be shorter than if the flanking cysteineresidues are linked to cysteine residues separated in primary sequence.Otherwise, particular amino acid substitutions can be introduced toconstrain a longer RGD-containing loop into an optimal conformation forhigh affinity integrin binding. The knottin polypeptide scaffolds usedherein may contain certain modifications made to truncate the nativeknottin, or to remove a loop or unnecessary cysteine residue ordisulfide bond.

Incorporation of integrin-binding sequences into a molecular (e.g.,knottin polypeptide) scaffold provides a framework for ligandpresentation that is more rigid and stable than linear or cyclic peptideloops. In addition, the conformational flexibility of small peptides insolution is high, and results in large entropic penalties upon binding.Such constructs have also been described in detail in InternationalPatent Publication WO 2016/025642, incorporated herein by reference inits entirety.

Incorporation of an integrin-binding sequence into a knottin polypeptidescaffold provides conformational constraints that are required for highaffinity integrin binding. Furthermore, the scaffold provides a platformto carry out protein engineering studies such as affinity or stabilitymaturation.

As used herein, the term “knottin protein” refers to a structural familyof small proteins, typically 25-40 amino acids, which bind to a range ofmolecular targets like proteins, sugars and lipids. Theirthree-dimensional structure is essentially defined by a peculiararrangement of three to five disulfide bonds. A characteristic knottedtopology with one disulfide bridge crossing the macro-cycle limited bythe two other intra-chain disulfide bonds, which was found in severaldifferent microproteins with the same cystine network, lent its name tothis class of biomolecules. Although their secondary structure contentis generally low, the knottins share a small triple-strandedantiparallel β-sheet, which is stabilized by the disulfide bondframework. Biochemically well-defined members of the knottin family,also called cystine knot proteins, include the trypsin inhibitor EETI-IIfrom Ecballium elaterium seeds, the neuronal N-type Ca²⁺ channel blockerco-conotoxin from the venom of the predatory cone snail Conusgeographus, agouti-related protein (AgRP, See Millhauser et al., “Loopsand Links: Structural Insights into the Remarkable Function of theAgouti-Related Protein,” Ann. N.Y. Acad. ScL, Jun. 1, 2003; 994(1):27-35), the omega agatoxin family, etc. A suitable agatoxin sequence[SEQ ID NO: 41] is given in U.S. Pat. No. 8,536,301, having a commoninventor with the present application. Other agatoxin sequences suitablefor use in the methods disclosed herein include, but are not limited toOmega-agatoxin-Aa4b (GenBank Accession number P37045) andOmega-agatoxin-Aa3b (GenBank Accession number P81744). Other knottinsequences suitable for use in the methods disclosed herein include,knottin [Bemisia tabaci] (GenBank Accession number FJ601218.1),Omega-lycotoxin (Genbank Accession number P85079), mu-O conotoxinMrVIA=voltage-gated sodium channel blocker (Genbank Accession numberAAB34917) and Momordica cochinchinensis Trypsin Inhibitor I (MCoTI-I) orII (MCoTI-II) (Uniprot Accession numbers P82408 and P82409,respectively).

Knottin proteins have a characteristic disulfide linked structure. Thisstructure is also illustrated in Gelly et al., “The KNOTTIN website anddatabase: a new information system dedicated to the knottin scaffold,”Nucleic Acids Research, 2004, Vol. 32, Database issue D156-D159. Atriple-stranded β-sheet is present in many knottins. The spacing betweencysteine residues is important, as is the molecular topology andconformation of the integrin-binding loop.

The term “molecular scaffold” means a polymer having a predefinedthree-dimensional structure, into which an integrin-binding loop isincorporated, such as an RGD peptide sequence as described herein. Theterm “molecular scaffold” has an art-recognized meaning (in othercontexts), which is also intended here. For example, a review by Skerra,“Engineered protein scaffolds for molecular recognition,” J. Mol.Recognit. 2000; 13: 167-187 describes the following scaffolds: singledomains of antibodies of the immunoglobulin superfamily, proteaseinhibitors, helix-bundle proteins, disulfide-knotted peptides andlipocalins. Guidance is given for the selection of an appropriatemolecular scaffold.

The term “knottin polypeptide scaffold” refers to a knottin proteinsuitable for use as a molecular scaffold, as described herein.Characteristics of a desirable knottin polypeptide scaffold forengineering include 1) high stability in vitro and in vivo, 2) theability to replace amino acid regions of the scaffold with othersequences without disrupting the overall fold, 3) the ability to createmultifunctional or bispecific targeting by engineering separate regionsof the molecule, and 4) a small size to allow for chemical synthesis andincorporation of non-natural amino acids if desired. Scaffolds derivedfrom human proteins are favored for therapeutic applications to reducetoxicity or immunogenicity concerns, but are not always a strictrequirement. Other scaffolds that have been used for protein designinclude fibronectin (Koide et al., 1998), lipocalin (Beste et al.,1999), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Hufton etal, 2000), and tendamistat (McConnell and Hoess, 1995; Li et al, 2003).While these scaffolds have proved to be useful frameworks for proteinengineering, molecular scaffolds such as knottins have distinctadvantages: their small size and high stability.

As used herein, the term “NOD201” refers to an integrin-bindingpolypeptide-Fc fusion comprising the following sequence:GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130; 2.5F peptide) andhaving no linker between the 2.5F peptide and the Fc domain. In someembodiments, the Fc domain is from IgG1, IgG2, IgG3, or IgG4 and can bemouse or human derived.

As used herein, the term “NOD201modK” refers to an integrin-bindingpolypeptide-Fc fusion comprising the following sequence:GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131; 2.5FmodK peptide) andhaving no linker between the 2.5FmodK peptide and the Fc domain. In someembodiments, the Fc domain is from IgG1, IgG2, IgG3, or IgG4 and can bemouse or human derived.

As used herein, the term “NOD203” refers to an integrin-bindingpolypeptide-Fc fusion comprising the following sequence:GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132; 2.5F peptide) andhaving a Gly4Ser linker between the 2.5F peptide and the Fc domain. Insome embodiments, the Fc domain is from IgG1, IgG2, IgG3, or IgG4 andcan be mouse or human derived.

As used herein, the term “NOD203modK” refers to an integrin-bindingpolypeptide-Fc fusion comprising the following sequence:GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133; 2.5FmodK peptide)and having a Gly4Ser linker between the 2.5FmodK peptide and the Fcdomain. In some embodiments, the Fc domain is from IgG1, IgG2, IgG3, orIgG4 and can be mouse or human derived.

As used herein, the term “NOD204” refers to an integrin-bindingpolypeptide-FC fusion comprising the following sequence:GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134; 2.5Fpeptide) and having a Gly4Ser3 linker between the 2.5F peptide and theFc domain. In some embodiments, the Fc domain is from IgG1, IgG2, IgG3,or IgG4 and can be mouse or human derived.

As used herein, the term “NOD204modK” refers to an integrin-bindingpolypeptide-FC fusion comprising the following sequence:CPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135; 2.5FmodKpeptide) and having a Gly4Ser3 linker between the 2.5FmodK peptide andthe Fc domain. In some embodiments, the Fc domain is from IgG1, IgG2,IgG3, or IgG4 and can be mouse or human derived.

As used herein, the term “AgRP” means PDB entry 1HYK. Its entry in theKnottin database is SwissProt AGRP HUMAN, where the full-length sequenceof 129 amino acids may be found. It comprises the sequence beginning atamino acid 87. An additional G is added to this construct. It alsoincludes a CI 05 A mutation described in Jackson, et al. 2002Biochemistry, 41, 7565, as well as International Patent Publication WO2016/025642, incorporated by reference in its entirety; bold andunderlined portion, from loop 4, is replaced by the RGD sequencesdescribed herein. Loops 1 and 3 are shown between brackets.

As used herein, “integrin-binding polypeptide-Fc fusion” is usedinterchangeably with “knottin-Fc” and refers to an integrin-bindingpolypeptide that includes an integrin-binding amino acid sequence withina knottin polypeptide scaffold and is operably linked to an Fc domain.In some embodiments, the Fc domain is fused to the N-terminus of theintegrin-binding polypeptide. In some embodiments, the Fc domain isfused to the C-terminus of the integrin-binding polypeptide. In someembodiments, the Fc domain is operably linked to the integrin-bindingpolypeptide via a linker.

As used herein, the term “Fc region” refers to the portion of a nativeimmunoglobulin formed by the respective Fc domains (or Fc moieties) ofits two heavy chains. As used herein, the term “Fc domain” refers to aportion of a single immunoglobulin (Ig) heavy chain wherein the Fcdomain does not comprise an Fv domain. As such, an Fc domain can also bereferred to as “Ig” or “IgG.” In certain embodiments, an Fc domainbegins in the hinge region just upstream of the papain cleavage site andends at the C-terminus of the antibody. Accordingly, a complete Fcdomain comprises at least a hinge domain, a CH₂ domain, and a CH₃domain. In certain embodiments, an Fc domain comprises at least one of:a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH₂domain, a CH₃ domain, a CH₄ domain, or a variant, portion, or fragmentthereof. In other embodiments, an Fc domain comprises a complete Fcdomain (i.e., a hinge domain, a CH₂ domain, and a CH₃ domain). In oneembodiment, an Fc domain comprises a hinge domain (or portion thereof)fused to a CH₃ domain (or portion thereof). In another embodiment, an Fcdomain comprises a CH₂ domain (or portion thereof) fused to a CH₃ domain(or portion thereof). In another embodiment, an Fc domain consists of aCH₃ domain or portion thereof. In another embodiment, an Fc domainconsists of a hinge domain (or portion thereof) and a CH₃ domain (orportion thereof). In another embodiment, an Fc domain consists of a CH₂domain (or portion thereof) and a CH₃ domain. In another embodiment, anFc domain consists of a hinge domain (or portion thereof) and a CH₂domain (or portion thereof). In one embodiment, an Fc domain lacks atleast a portion of a CH₂ domain (e.g., all or part of a CH₂ domain). AnFc domain herein generally refers to a polypeptide comprising all orpart of the Fc domain of an immunoglobulin heavy-chain. This includes,but is not limited to, polypeptides comprising the entire CH₁, hinge,CH₂, and/or CH₃ domains as well as fragments of such peptides comprisingonly, e.g., the hinge, CH₂, and CH₃ domain. The Fc domain may be derivedfrom an immunoglobulin of any species and/or any subtype, including, butnot limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. A human IgG1 constant region can be found at Uniprot P01857and in FIG. 1. The Fc domain of human IgG1 with a deletion of the upperhinge region can be found in Table 2, SEQ ID NO: 3 from InternationalPatent Publication No. WO 2016/025642. The Fc domain encompasses nativeFc and Fc variant molecules. As with Fc variants and native Fc's, theterm Fc domain includes molecules in monomeric or multimeric (e.g.,dimeric) form, whether digested from whole antibody or produced by othermeans. The assignment of amino acid residue numbers to an Fc domain isin accordance with the definitions of Kabat. See, e.g., Sequences ofProteins of Immunological Interest (Table of Contents, Introduction andConstant Region Sequences sections), 5^(th) edition, Bethesda, Md.; NIHvol. 1:647-723 (1991); Kabat et al., “Introduction” Sequences ofProteins of Immunological Interest, US Dept of Health and HumanServices, NIH, 5th edition, Bethesda, Md. vol. 1:xiii-xcvi (1991);Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al, Nature342:878-883 (1989), each of which is herein incorporated by referencefor all purposes. With regard to the integrin-binding polypeptide-Fcfusions described herein, any Fc domain from any IgG as described hereinor known can be employed as part of the Fc fusion, including mouse,human and variants thereof, such as hinge deleted (EPKSC deleted; see,SEQ ID NO: 3 from International Patent Publication No. WO 2016/025642).

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain exemplary embodiments, the Fc domainhas increased effector function (e.g., FcγR binding).

The Fc domains of a polypeptide of the invention may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH₂ and/or CH₃ domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another peptide may have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants, in the context of IL-2 or a knottin protein,necessarily have less than 100% sequence identity or similarity with thestarting IL-2 or knottin protein. In some embodiments, the variant willhave an amino acid sequence from about 75% to less than 100% amino acidsequence identity or similarity with the amino acid sequence of thestarting polypeptide, more preferably from about 80% to less than 100%,more preferably from about 85% to less than 100%, more preferably fromabout 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%) and in some embodiments from about 95% to less than 100%,e.g., over the length of the variant molecule.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide comprising IL-2 or a variant thereof,for use in extended-PK IL-2 consists of, consists essentially of, orcomprises an amino acid sequence selected from SEQ ID Nos: 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 from InternationalPatent Publication No. WO 2016/025642 (copied below). In an embodiment,a polypeptide includes an amino acid sequence at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence selected from SEQID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35from International Patent Publication No. WO 2016/025642 (copied below).In an embodiment, a polypeptide includes a contiguous amino acidsequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguousamino acid sequence selected from SEQ ID Nos: 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, and 35 from International PatentPublication No. WO 2016/025642 (copied below). In an embodiment, apolypeptide includes an amino acid sequence having at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200,300, 400, or 500 (or any integer within these numbers) contiguous aminoacids of an amino acid sequence selected from SEQ ID Nos: 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 from InternationalPatent Publication No. WO 2016/025642 (copied below).

In an embodiment, the peptides are encoded by a nucleotide sequence.Nucleotide sequences can be useful for a number of applications,including: cloning, gene therapy, protein expression and purification,mutation introduction, DNA vaccination of a host in need thereof,antibody generation for, e.g., passive immunization, PCR, primer andprobe generation, and the like. In an embodiment, the nucleotidesequence of the invention comprises, consists of, or consistsessentially of, a nucleotide sequence of IL-2, or a variant thereof,selected from SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, and 34 from International Patent Publication No. WO2016/025642 (copied below). In an embodiment, a nucleotide sequenceincludes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleotide sequence set forth in SEQ ID Nos: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 from InternationalPatent Publication No. WO 2016/025642 (copied below). In an embodiment,a nucleotide sequence includes a contiguous nucleotide sequence at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotidesequence set forth in SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, and 34 from International Patent Publication No. WO2016/025642 (copied below). In an embodiment, a nucleotide sequenceincludes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or500 (or any integer within these numbers) contiguous nucleotides of anucleotide sequence set forth in SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, and 34 from International PatentPublication No. WO 2016/025642 (copied below).

TABLE 1 Sequence Summary SEQ ID NO Description Sequence 1 HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS IgG1SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL constantGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE regionQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP (amino acidSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV sequence)DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 HumanEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK IgG1 FcFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE domainKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK (aminoTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK acid sequence) 3Human DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVIgG1 Fc DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKdomain AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV(amino LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK acid sequence)Deletion (AEPKSC) Upper Hinge 4 Mouse GCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCA IL-2GCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGA (nucleicGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTAC acidTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACC sequence)TCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAA 5 MouseAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFY IL-2LPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTWKLKGSDNTF (aminoECQFDDESATWDFLRRWIAFCQSIISTSPQ acid sequence) 6 QQ6210GCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCA (nucleicGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAACTCCTGA acidGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTAC sequence)TTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAA 7 QQ6210APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKFY (aminoLPEQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTF acidECQFDDEPATWDFLRRWIAFCQSIISTSPQ sequence) 8 E76AGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCA (nucleicGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGA acidGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTAC sequence)TTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGCTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAA 9 E76AAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFY (aminoLPKQATELKDLQCLEDALGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTF acidECQFDDESATWDFLRRWIAFCQSIISTSPQ sequence) 10 E76GGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCA (nucleicGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGA acidGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTAC sequence)TTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGGTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAA 11 E76GAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFY (aminoLPKQATELKDLQCLEDGLGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTF acidECQFDDESATWDFLRRWIAFCQSIISTSPQ sequence) 12 D265AATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCACGATG Fc/FlagTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCAT (nucleicGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAG acidGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGCCGTGAGCGA sequence)GGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTC (C-terminalAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCC flag tagATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGC isCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCAC underlined)AGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGTGGCGGATCTGACTACAAGGACGACGATGACAAGTGATAA 13 D265AMRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIK Fc/FlagDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP (aminoIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLT acidCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFAC sequence)SWHEGLHNHLTTKTISRSLGKGGGSDYKDDDDK (C-terminal flag tag is underlined) 14D265A ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCACGATG Fc/wtTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCAT mIL-2GCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAG (nucleicGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGCCGTGAGCGA acidGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTC sequence)AGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCC (C-terminalATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGC 6x hisCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCAC tag isAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACC underlined)TGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATAA 15 D265AMRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIK Fc/wDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP t mIL-2IQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLT (aminoCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFAC acidSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLM sequence)DLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF (C-terminalQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQHH 6x hisHHHH** tag is underlined) 16 D265A FcATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCACGATG QQ6210TGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCAT (nucleicGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAG acidGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGCCGTGAGCGA sequence)GGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTC (C-terminalAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCC6x his tag isATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGC underlined)CCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAACTCCTGAGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATAA 17 D265A Fc/MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIK QQ6210DVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP (aminoIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLT acidCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFAC sequence)SVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLM (C-terminalDLQELLSRMEDHRNLRLPRMLTFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSKSF 6x hisQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSPQHH tag is HHHHunderlined) 18 D265A Fc/ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCACGATG E76ATGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCAT (nucleicGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAG acidGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGCCGTGAGCGA sequence)GGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTC (C-terminalAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCC 6x hisATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGC tag isCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCAC underlined)AGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGCTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATAA 19 D265A Fc/MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIK E76ADVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRWSALP (aminoIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLT acidCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFAC sequence)SVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLM (C-terminalDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLOCLEDALGPLRHVLDLTQSKSF 6x hisQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQHH tag is HHHHunderlined) 20 D265A Fc/ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCACGATG E76GTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCAT (nucleicGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAG acidGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGCCGTGAGCGA sequence)GGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTC (C-terminalAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCC 6x hisATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGC tag isCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCAC underlined)AGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGGTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATAA 21 D265A Fc/MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIK E76GDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALP (aminoIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLT acidCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFAC sequence)SVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSSTAEAQQQQQOQQQQQQHLEQLLM (C-terminalDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDGLGPLRHVLDLTQSKSF 6x hisQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQHH tag is HHHHunderlined) 22 mIL-2 QQGCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCA 6.2-4GCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGA (nucleicGCAGGATGGAGGATTCCAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTAC acidTTGCCCAAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACC sequence)TCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGGCTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACGAGCCCTCAA 23 mIL-2 QQAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDSRNLRLPRMLTFKFY 6.2-4LPKQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTF (aminoECQFDDEPATVVGFLRRWIAFCQSIISTSPQ acid sequence) 24 mIL-2 QQGCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCA 6.2-8GCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGTAGGATGGAGG (nucleicATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAG acidGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGT sequence)TCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCGA 25 mIL-2 QQAPTSSSTSSSTAEAQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKFYLPKQ 6.2-8ATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQF (aminoDDEPATWDFLRRWIAFCQSIISTSPR acid sequence) 26 mIL-2 QQGCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCA 6.2-10GCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAACTCCTGA (nucleicGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTAC acidTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACC sequence)ACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAG 27 mIL-2 QQAPTSSSTSSSTAEAOQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKFY 6.2-10LPEQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTF (aminoECQFDDEPATVVDFLRRWIAFCQSIISTSPQ acid sequence) 28 mIL-2 QQGCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCA 6.2-11GCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGG (nucleicAGGATTCCAGGAACCTGAGACTCCCCAGAATGCTCACCTTCAAATTTTACTTGCCCGAG acidCAGGCCACAGAATTGAAAGATCTCCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCA sequence)AGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAG 29 mIL-2 QQAPTSSSTSSSTAEAQQQQQQQQQHLEQLLMDLQELLSRMEDSRNLRLPRMLTFKFYLPE 6.2-11QATELKDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQ (amino acidFDDEPATVVDFLRRWIAFCQSIISTSPQ sequence) 30 mIL-2 QQGCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCA 6.2-13GCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGA (nucleicGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTAC acidTTGCCCGAGCAGGCCACAGAATTGAAAGATCTCCAGTGCCTAGAAGATGAACTTGAACC sequence)TCTGCGGCAGGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAG 31 mIL-2 QQAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKFY 6.2-13LPEQATELKDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTF (amino acidECQFDDEPATVVDFLRRWIAFCQSIISTSPQ sequence) 32 Full lengthATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAG human IL-2TGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGG (nucleicATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATG acidCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCT sequence)AGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA 33 Full lengthMYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM human IL-2LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKG (amino acidSETTFMCEYADETATIVEFLNRWITFCQSIISTLT sequence) 34 Human IL-2GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGA withoutTTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGC signalTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTA peptideGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCA (nucleicCTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGAT acidCTGAAACAACATTCATGTGTAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGA sequence)ACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA 35 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL withoutEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL signalNRWITFCQSIISTLT peptide (amino acid sequence) 36 HumanMDMRVPAQLLGLLLLWLPGARCADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF serumEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE albuminPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPEL (amino acidLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS sequence)SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGS 37 MatureDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESA HSA (aminoENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRP acidEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC sequence)LLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGS 38 HumanATGGATATGCGGGTGCCTGCTCAGCTGCTGGGACTGCTGCTGCTGTGGCTGCCTGGGGC serumTAGATGCGCCGATGCTCACAAAAGCGAAGTCGCACACAGGTTCAAAGATCTGGGGGAGG albuminAAAACTTTAAGGCTCTGGTGCTGATTGCATTCGCCCAGTACCTGCAGCAGTGCCCCTTT (nucleicGAGGACCACGTGAAACTGGTCAACGAAGTGACTGAGTTCGCCAAGACCTGCGTGGCCGA acidCGAATCTGCTGAGAATTGTGATAAAAGTCTGCATACTCTGTTTGGGGATAAGCTGTGTA sequence)CAGTGGCCACTCTGCGAGAAACCTATGGAGAGATGGCAGACTGCTGTGCCAAACAGGAACCCGAGCGGAACGAATGCTTCCTGCAGCATAAGGACGATAACCCCAATCTGCCTCGCCTGGTGCGACCTGAGGTGGACGTCATGTGTACAGCCTTCCACGATAATGAGGAAACTTTTCTGAAGAAATACCTGTACGAAATCGCTCGGAGACATCCTTACTTTTATGCACCAGAGCTGCTGTTCTTTGCCAAACGCTACAAGGCCGCTTTCACCGAGTGCTGTCAGGCAGCCGATAAAGCTGCATGCCTGCTGCCTAAGCTGGACGAACTGAGGGATGAGGGCAAGGCCAGCTCCGCTAAACAGCGCCTGAAGTGTGCTAGCCTGCAGAAATTCGGGGAGCGAGCCTTCAAGGCTTGGGCAGTGGCACGGCTGAGTCAGAGATTCCCAAAGGCAGAATTTGCCGAGGTCTCAAAACTGGTGACCGACCTGACAAAGGTGCACACCGAATGCTGTCATGGCGACCTGCTGGAGTGCGCCGACGATCGAGCTGATCTGGCAAAGTATATTTGTGAGAACCAGGACTCCATCTCTAGTAAGCTGAAAGAATGCTGTGAGAAACCACTGCTGGAAAAGTCTCACTGCATTGCCGAAGTGGAGAACGACGAGATGCCAGCTGATCTGCCCTCACTGGCCGCTGACTTCGTCGAAAGCAAAGATGTGTGTAAGAATTACGCTGAGGCAAAGGATGTGTTCCTGGGAATGTTTCTGTACGAGTATGCCAGGCGCCACCCAGACTACTCCGTGGTCCTGCTGCTGAGGCTGGCTAAAACATATGAAACCACACTGGAGAAGTGCTGTGCAGCCGCTGATCCCCATGAATGCTATGCCAAAGTCTTCGACGAGTTTAAGCCCCTGGTGGAGGAACCTCAGAACCTGATCAAACAGAATTGTGAACTGTTTGAGCAGCTGGGCGAGTACAAGTTCCAGAACGCCCTGCTGGTGCGCTATACCAAGAAAGTCCCACAGGTGTCCACACCCACTCTGGTGGAGGTGAGCCGGAATCTGGGCAAAGTGGGGAGTAAATGCTGTAAGCACCCTGAAGCCAAGAGGATGCCATGCGCTGAGGATTACCTGAGTGTGGTCCTGAATCAGCTGTGTGTCCTGCATGAAAAAACACCTGTCAGCGACCGGGTGACAAAGTGCTGTACTGAGTCACTGGTGAACCGACGGCCCTGCTTTAGCGCCCTGGAAGTCGATGAGACTTATGTGCCTAAAGAGTTCAACGCTGAGACCTTCACATTTCACGCAGACATTTGTACCCTGAGCGAAAAGGAGAGACAGATCAAGAAACAGACAGCCCTGGTCGAACTGGTGAAGCATAAACCCAAGGCCAC/QAAGAGCAGCTGAAGGCTGTCATGGACGATTTCGCAGCCTTTGTGGAAAAATGCTGTAAGGCAGACGATAAGGAGACTTGCTTTGCCGAGGAAGGAAAGAAACTGGTGGCTGCATCCCAGGCAGCTCTGGGACTGGGAGGAGGATCTGCCCCTACCTCAAGCTCCACTAAGAAAACCCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATGATTCTGAACGGGATCAACAATTACAAAAATCCAAAGCTGACCCGGATGCTGACATTCAAGTTTTATATGCCCAAGAAAGCCACAGAGCTGAAACACCTGCAGTGCCTGGAGGAAGAGCTGAAGCCTCTGGAAGAGGTGCTGAACCTGGCCCAGAGCAAGAATTTCCATCTGAGACCAAGGGATCTGATCTCCAACATTAATGTGATCGTCCTGGAACTGAAGGGATCTGAGACTACCTTTATGTGCGAATACGCTGACGAGACTGCAACCATTGTGGAGTTCCTGAACAGATGGATCACCTTCTGCCAGTCCATCATTTCTACTCTGACAGGCGGGG GGAGC 39EETI-II GC PRILMR CKQDSDCLAGCVCGPNGFCG from Knottin Database 40AgRP from GCVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCR-KLGTAMNPCSRT KnottinDatabase “-” indicates where mini protein can be formed 41 OmegaEDN--CIAEDYGKCTWGGTKCCRGRPCRC SMIGTN CECTPRLIMEGLSFA agatoxin fromKnottin Database “-” indicates where mini protein can be formed 42EETI-II GCXXXRGDXXXXXCKQDSDCLAGCVCGPNGFCG Librarv 43 EETI-IIGCXXXRGDXXXXXCSQDSDCLAGCVCGPNGFCG K15S Mutation Library 44 2.5F-GGTTGTCCAAGACCAAGAGGTGATAATCCACCATTGACTTGTTCTCAAGATTCTGATTG (K15S)TTTGGCTGGTTGTGTTTGTGGTCCAAATGGTTTTTGTGGTGGTCGACTAGAGCCCAGAG mIgG2aFcTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCA NucleicGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCAT AcidGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAG SequenceACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAA 45 2.5F-GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGEPRVPITQNPCPPLKECPPCAAPDLL (K15S)GGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE mIgG2aFcDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPP AminoPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRV AcidQKSTWERGSLFACSWHEGLHNHLTTKTISRSLGK Sequence 46 2.5D-GGTTGTCCACAAGGCAGAGGTGATTGGGCTCCAACTTCTTGTTCTCAAGATTCTGATTG (K15S)TTTGGCTGGTTGTGTTTGTGGTCCAAATGGTTTTTGTGGTGGTCGACTAGAGCCCAGAG mIgG2aFcTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCA NucleicGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCAT AcidGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAG SequenceACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAA 47 2.5D-GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGEPRVPITQNPCPPLKECPPCAAPDLL (K15S)GGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE mIgG2aFcDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPP AminoPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRV AcidQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK Sequence 48 2.5F-GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGEPKSCDKTHTCPPCPAPELLGGPSVF (K15S)LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY hIgG1FcRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT AminoKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ AcidQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence 49 2.5F-GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGDKTHTCPPCPAPELLGGPSVFLFPPK (K.15S)PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV hIgG1FcLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS Fc UpperLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF HingeSCSVMHEALHNHYTQKSLSLSPGK Deletion (AEPKSC) Amino Acid Sequence 50 2.5D-GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGEPKSCDKTHTCPPCPAPELLGGPSVF (K15S)LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY hIgG1FcRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT AminoKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ AcidQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence 51 2.5D-GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGDKTHTCPPCPAPELLGGPSVFLFPPK (K15S)PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV hIgG1FcLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS Fc UpperLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF HingeSCSVMHEALHNHYTQKSLSLSPGK Deletion (AEPKSC) Amino Acid Sequence 52 hPD-1MQIPQAPWPVVWAVLQLGWRPGWFLDSPDPWNPPTFFPALLVVTEGDNATFTCSFSNTS amino ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSG acidTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLL sequenceGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL 53 hPD-L1MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEM aminoEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY acidGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSG sequenceKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLC LGVALTFIFR LRKGRMMDVKKCGIQDTNSK KQSDTHLEET 54 hCTLA-4MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASS aminoRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM acidMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY sequenceLGIGNGTQIY VIDPEPCPDS DFLLWILAAVSSGLFFYSFLLTAVSLSKML KKRSPLTTGVYVKMPPTEPE CEKQFQPYFI PIN 55 hLAG-3MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRA aminoGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQP acidRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPG sequenceSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFGFHLWRRQWRPRRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL 56 hTIM-3MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACP aminoVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIM acidNDEKFNLKLVIKPAKVTPAPTR QRDFTAAFPR MLTTRGHGPA ETOTLGSLPD sequenceINLTQISTLA NELRDSRLANDLRDSGATIRGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFA MP 57 hB7-H3MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPWALVGTDATLCC aminoSFSPEPGFSLQLNLIWQLT DTKQLVHSFA EGQDQGSAYA acidNRTALFPDLLAQGNASLRLQRVRVADEGSFCFVSIRDFGSAAVSLQVAA sequencePYSKPSMTLE PNKDLRPGDT VTITCSSYQG YPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPWALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRV RVADEGSFTC FVSIRDFGSA AVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRWLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPIKHSDSKEDDGQEIA 58 hB7-H4MASLGQILFWSIISIIIILAGAIALIIGFGISAFSMPEVNVDYNAS SETLRCEAPR aminoWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKWSVLYN VTINNTYSCM acidIENDIAKATGDIKVTESEIKRRSHLQLLNS KASLCVSSFFAISWALLPLSPYLMLK sequence 145TIGIT MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST isoform 1TAQVTQVNWE QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV aminoNDTGEYFCIY HTYPDGTYTG RIFLEVLESS VAEHGARFQI PLLGAMAATL acidVVICTAVIVV VALTRKKKAL RIHSVEGDLR RKSAGQEEWS PSAPSPPGSC sequenceVQAEAAPAGL CGEQRGEDCA ELHDYFNVLS YRSLGNCSFF TETG Q495A1-1 146 TIGITMRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST isoform 1TAQVTQVNWE QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV aminoNDTGEYFCIY HTYPDGTYTG RIFLEVLESS VAEHGARFQI PLLGAMAATL acidVVICTAVIVV VALTRKFVCF sequence Q495A1-2 147 4-MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP IBB/CD137NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS aminoMCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG acidTKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL sequenceFLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE Q07011-1 GGCEL148 IFNα MASPFALLMV LVVLSCKSSC SLGCDLPETH SLDNRRTLML LAQMSRISPS aminoSCLMDRHDFG FPQEEFDGNQ FQKAPAISVL HELIQQIFNL FTTKDSSAAW acidDEDLLDKFCT ELYQQLNDLE ACVMQEERVG ETPLMNADSI LAVKKYFRRI sequenceTLYLTEKKYS PCAWEWRAE IMRSLSLSTN LQERLRRKE P01562 149 GITRMAQHGAMGAF RALCGLALLC ALSLGQRPTG GPGCGPGRLL LGTGTDARCC amino acidRVHTTRCCRD YPGEECCSEW DCMCVQPEFH CGDPCCTTCR HHPCPPGQGV sequenceQSQGKFSFGF QCIDCASGTF SGGHEGHCKP WTDCTQFGFL TVFPGNKTHN Q9Y5U5AVCVPGSPPA EPLGWLTVVL LAVAACVLLL TSAQLGLHIW QLRSQCMWPRETQLLLEVPP STEDARSCQF PEEERGERSA EEKGRLGDLW V 150 OX40MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN amino acidGMVSRCSRSQ NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT sequenceATQDTVCRCR AGTQPLDSYK PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA P43489GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQGPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL RRDQRLPPDAHKPPGGGSFR TPIQEEQADA HSTLAKI 151 CD40MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD amino acidCTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD sequenceTICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF P25942FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDWCGPQD RLRALVVIPIIFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ 152 ICOSMKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ amino acidFKMQLLKGGQ ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD sequenceHSHANYYFCN LSIFDPPPFK VTLTGGYLHI YESQLCCQLK FWLPIGCAAF Q9Y6W8VVVCILGCIL ICWLTKKKYS SSVHDPNGEY MFMRAVNTAK KSRLTDVTL 153 CD28MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE amino acidFRASLHKGLD SAVEVCWYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ sequenceNLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS P10747KPFWVLVWG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS 154 IFNαMALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRKISLF amino acidSCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD sequenceETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT P01563LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE

In one embodiment, an integrin-binding polypeptide or a variant thereof,consists of, consists essentially of, or comprises an amino acidsequence selected from SEQ ID NOs: 59-135. In an embodiment, apolypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence selected from SEQ IDNos: 59-135. In an embodiment, a polypeptide includes a contiguous aminoacid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to acontiguous amino acid sequence selected from SEQ ID Nos: 59-135. In anembodiment, a polypeptide includes an amino acid sequence having atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers)contiguous amino acids of an amino acid sequence selected from SEQ IDNOs: 59-135.

TABLE 2 Integrin Binding Knottin Sequences SEQ ID PeptideSequence (RGD motif is underlined NO: Identifier Scaffoldwith flanking residues) 59 1.4A EETI-II GC AEPRGDMPWTWCKQDSDCLAGCVCGPNGFCG 60 1.4B EETI-II GC VGGRGDWSPKW CKGDSDCPAGCVCGPNGFCG61 1.4C EETI-II GC  AELRGDRSYPE  CKQDSDCLAGCVCGPNGFCG 62 1.4E EETI-II GG RLPRGDVPRPH  CKQDSDCQAGCVCGPNGFCG 63 1.4H EETI-II GC  YPLRGDNPYAA CKQDSDCRAGCVCGPNGFCG 64 1.5B EETI-II GC  TIGRGDWAPSE CKQDSDCLAGCVCGPNGFCG 65 l.SF EETI-II GC  HPPRGDNPPVT CKQDSDCLAGCVCGPNGFCG 66 2.3A EETI-II GC  PEPRGDNPPPS CKQDSDCRAGCVCGPNGFCG 67 2.3B EETI-II GC  LPPRGDNPPPS CKQDSDCQAGCVCGPNGFCG 68 2.3C EETI-II GC HLGRGDWAPVG CKQDSDCPAGCVCGPNGFCG69 2.3D EETI-II GC  NVGRGDWAPSE CKQDSDCPAGCVCGPNGFCG 70 2.3E EETI-II GC FPGRGDWAPSS CKQDSDCRAGCVCGPNGFCG 71 2.3F EETI-II GC  PLPRGDNPPTECKQDSDCQAGCVCGPNGFCG 72 2.3G EETI-II GC  SEARGDNPRLSCKQDSDCRAGCVCGPNGFCG 73 2.3H EETI-II GC LLGRGDWAPEA CKQDSDCRAGCVCPNGFCG74 2.31 EETI-II GC HVGRGDWAPLK CKQDSDCQAGCVCGPNGFCG 75 2.3J EETI-II GC VRGRGDWAPPS CKQDSDCPAGCVCGPNGFCG 76 2.4A EETI-II GC LGGRGDWAPPACKQDSDCRAGCVCGPNGFCG 77 2.4C EETI-II GC  FVGRGDWAPLTCKQDSDCQAGCVCGPNGFCG 78 2.4D EETI-II GC  PVGRGDWSPASCKQDSDCRAGCVCGPNGFCG 79 2.4E EETI-II GC  PRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG 80 2.4F EETI-II GC  YQGRGDWSPSSCKQDSDCPAGCVCGPNGFCG 81 2.4G EETI-II GC  APGRGDWAPSECKQDSDCQAGCVCGPNGFCG 82 2.4J EETI-II GC  VQGRGDWSPPSCKQDSDCPAGCVCGPNGFCG 83 2.SA EETI-II GC  HVGRGDWAPEECKQDSDCQAGCVCGPNGFCG 84 2.SC EETI-II GC  DGGRGDWAPPACKQDSDCRAGCVCGPNGFCG 85 2.5D EETI-II GC  PQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG 86 2.SF EETI-II GC PRPRGDNPPLT CKQDSDCLAGCVCGPNGFCG87 2.5D K15S EETI-II GC PQGRGDWAPTS CSQDSDCLAGCVCGPNGFCG Mutant 882.5F K15S EETI-II GC PRPRGDNPPLT CSQDSDCLAGCVCGPNGFCG Mutant 89 2.SHEETI-II GC PQGRGDWAPEW CKQDSDCPAGCVCGPNGFCG 90 2.SJ EETI-II GCPRGRGDWSPPA CKQDSDCQAGCVCGPNGFCG 91 3A AgRp GCVRLHESCLGQQVPCCDPAATCYCVVRGDWRKR C YCR 92 3B AqRp GCVRLHESCLGQQVPCCDPAATCYC EERGDMLEK CYCR 933C AgRp GCVRLHESCLGQQVPCCDPAATCYC ETRGDGKEKCYCR 94 3D AgRpGCVRLHESCLGQQVPCCDPAATCYO QWRGDGDVK C YCR 95 3E AgRpGCVRLHESCLGQQVPCCDPAATCYC SRRGDMRER C YCR 96 3F AgRpGCVRLHESCLGQQVPCCDPAATCYC QYRGDGMKH C YCR 97 3G AgRpGCVRLHESCLGQQVPCCDPAATCYC TGRGDTKVL CYCR 98 3H AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDMKRR C YCR 99 3I AgRpGCVRLHESCLGQQVPCCDPAATCYC TGRGDVRMN CYCR 100 3J AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDGMSK C YCR 101 4A AgRpGCVRLHESCLGQQVPCCDPAATCYC RGRGDMRRE C YCR 102 4B AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDVKVN CYCR 103 4C AgRpGCVRLHESCLGQQVPCCDPAATCYC VGRGDEKMS C YCR 104 4D AgRpGCVRLHESCLGQQVPCCDPAATCYC VSRGDMRKR C YCR 105 4E AgRpGCVRLHESCLGQQVPCCDPAATCYC ERRGDSVKK CYCR 106 4F AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDTRRR CYCR 107 4G AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDWRR CYCR 108 4H AgRpGCVRLHESCLGQQVPCCDPAATCYC KGRGDNKRK C YCR 109 4I AgRpGCVRLHESCLGQQVPCCDPAXTCYC KGRGDVRRV CYCR 110 4J AgRpGCVRLHESCLGQQVPCCDPAATCYC VGRGDNKVK CYCR 111 5A AgRpGCVRLHESCLGQQVPCCDPAATCYC VGRGDNRLK CYCR 112 5B AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDGMKK C YCR 113 5C AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDMRRR C YCR 114 5D AgRpGCVRLHESCLGQQVPCCDPAATCYC QGRGDGDVK C YCR 115 5E AgRpGCVRLHESCLGQQVPCCDPAATCYC SGRGDNDLV CYCR 116 5F AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDGMIRC YCR 117 5G AgRpGCVRLHESCLGQQVPCCDPAATCYC SGRGDNDLV CYCR 118 5H AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDMKMK C YCR 119 5I AgRpGCVRLHESCLGQQVPCCDPAATCYC IGRGDVRRR CYCR 120 5J AgRpGCVRLHESCLGQQVPCCDPAATCYC EERGDGRKK CYCR 121 6B AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDRDMK C YCR 122 6C AgRpGCVRLHESCLGQQVPCCDPAATCYC TGRGDEKLR CYCR 123 6E AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDGNRR CYCR 124 6F AgRpGCVRLHESCLGQQVPCCDPAATCYC ESRGDWRK CYCR 125 7C AgRpGCVRLHESCLGQQVPCCDPAATCYC YGRGDNDLR C YCR

TABLE 3 Integrin Binding Polypeptide Sequences, Signal Sequences, Linkers, Fc fusions SEQ Peptide ID Identifier NO: Scaffold Sequence 130NOD201-2.SF GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG 131 NOD201modK-GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG 2.SFmodK 132 NOD203-2.SFGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS w/GGGGS 133 NOD203modKGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS 2.5FmodK w/GGGGS 134 NOD204-2.5FGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS w/GGGGSGGGGSGG GGS 135NOD204modK- GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS 2.5FmodK w/GGGGSGGGGSGGGG S 136 Linker (short) GGGGS (linker for use with anysequnces disclosed herein) 137 Linker (long) GGGGSGGGGSGGGGS (linker foruse with any sequnces disclosed herein) 138 Signal MTRLTVLALLAGLLASSRsequence (signal peptide A) (signal peptide for use with any sequncesdisclosed herein, including SEQ ID Nos: 139, 140, 141, 142, and 143) 139NOD201 (human GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGEPKSSDKTHTCPPCPAFc; no linker) PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG140 NOD201X GCVTGRDGSPASSCSQDSDCLAGCVCGPNGFCGEPKSSDKTHTCPPCPA (controlPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD sequence-GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP NOD201 withAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA scrambled seq.VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV human Fc; noMHEALHNHYTQKSLSLSPG linker) Theoretical pI/Mw: 6.19/ 58065.44 141NOD201M GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGEPRVPITQNPCPPLKE (NOD201 withCPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQ murine FcISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV domain; noNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGF linker)LPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGS TheoreticalLFACSWHEGLHNHLTTKTISRSLG pI/Mw: 6.34/ 59357.92 Ext. coefficient 60525Abs 0.1% (= 1 g/l) 1.020, assuming all pairs of Cys residues formcystines 142 NOD203 GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSEPKSSDKTHTCcomplete PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF (Gly₄SerNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS linker)NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 143 NOD204GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGSE completePKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD ([Gly₄Ser]₃VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL linker)NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

TABLE 4 Exemplary IgG sequenes: SEQ ID NO: Name Sequence 126 IgG1ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS  60GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 120PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 180STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 240LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300QQGNVFSCSV MHEALHNHYT QKSLSLSPGK                                  330127 IgG2ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS  60GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP APPVAGPSVF 120LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTFR 180VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN 240QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN 300VFSCSVMHEA LHNHYTQKSL SLSPGK                                      326128 IgG3ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS  60GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRVEL KTPLGDTTHT CPRCPEPKSC 120DTPPPCPRCP EPKSCDTPPP CPRCPEPKSC DTPPPCPRCP APELLGGPSV FLFPPKPKDT 180LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK PREEQYNSTF RVVSVLTVLH 240QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK 300GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 360ALHNRFTQKS LSLSPGK                                                377129 IgG4ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS  60GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP APEFLGGPSV 120FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 180RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK 240NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 300NVFSCSVMHE ALHNHYTQKS LSLSLGK                                     327

It will also be understood by one of ordinary skill in the art that theIL-2, extended-PK IL-2 or an integrin-binding polypeptide-Fc fusion usedherein may be altered such that they vary in sequence from the naturallyoccurring or native sequences from which they were derived, whileretaining the desirable activity of the native sequences. For example,nucleotide or amino acid substitutions leading to conservativesubstitutions or changes at “non-essential” amino acid residues may bemade. Mutations may be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis.

The polypeptides described herein (e.g., IL-2, extended-PK IL-2, PKmoieties, knottin, Fc, knottin-Fc, integrin-binding polypeptide-Fcfusion, and the like) may comprise conservative amino acid substitutionsat one or more amino acid residues, e.g., at essential or non-essentialamino acid residues. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, anonessential amino acid residue in a binding polypeptide is preferablyreplaced with another amino acid residue from the same side chainfamily. In another embodiment, a string of amino acids can be replacedwith a structurally similar string that differs in order and/orcomposition of side chain family members. Alternatively, in anotherembodiment, mutations may be introduced randomly along all or part of acoding sequence, such as by saturation mutagenesis, and the resultantmutants can be incorporated into binding polypeptides of the inventionand screened for their ability to bind to the desired target.

The “Programmed Death-1 (PD-1)” receptor refers to an immuno-inhibitoryreceptor belonging to the CD28 family. PD-1 is expressed predominantlyon previously activated T-cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. AAC51773 (SEQ ID NO: 52 fromInternational Publication No. WO 2016/025642).

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulatesT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7 (SEQ ID NO: 53 fromInternational Publication No. WO 2016/025642).

“Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4)” is a T cellsurface molecule and is a member of the immunoglobulin superfamily. Thisprotein downregulates the immune system by binding to CD80 and CD86. Theterm “CTLA-4” as used herein includes human CTLA-4 (hCTLA-4), variants,isoforms, and species homologs of hCTLA-4, and analogs having at leastone common epitope with hCTLA-4. The complete hCTLA-4 sequence can befound under GenBank Accession No. P16410 (SEQ ID NO: 54 fromInternational Publication No. WO 2016/025642):

“Lymphocyte Activation Gene-3 (LAG-3)” is an inhibitory receptorassociated with inhibition of lymphocyte activity by binding to MHCclass II molecules. This receptor enhances the function of Treg cellsand inhibits CD8+ effector T cell function. The term “LAG-3” as usedherein includes human LAG-3 (hLAG-3), variants, isoforms, and specieshomologs of hLAG-3, and analogs having at least one common epitope. Thecomplete hLAG-3 sequence can be found under GenBank Accession No. P18627(SEQ ID NO: 55 from International Publication No. WO 2016/025642).

“T-Cell Membrane Protein-3 (TIM-3)” is an inhibitory receptor involvedin the inhibition of lymphocyte activity by inhibition of T-cell andB-cell responses. Its ligand is galectin 9, which is upregulated invarious types of cancers. The term “TIM-3” as used herein includes humanTIM-3 (hTIM-3), variants, isoforms, and species homologs of hTIM-3, andanalogs having at least one common epitope. The complete hTIM-3 sequencecan be found under GenBank Accession No. Q8TDQO (SEQ ID NO: 56 fromInternational Publication No. WO 2016/025642).

The “B7 family” refers to inhibitory ligands with undefined receptors.The B7 family encompasses B7-H3 and B7-H4, both upregulated on tumorcells and tumor infiltrating cells. The complete hB7-H3 and hB7-H4sequence can be found under GenBank Accession Nos. Q5ZPR3 and AAZ17406(SEQ ID NOs: 57 and 58 from International Publication No. WO2016/025642) respectively.

“Vascular Endothelial Growth Factor (VEGF)” is a secreteddisulfide-linked homodimer that selectively stimulates endothelial cellsto proliferate, migrate, and produce matrix-degrading enzymes, all ofwhich are processes required for the formation of new vessels. Inaddition to being the only known endothelial cell specific mitogen, VEGFis unique among angiogenic growth factors in its ability to induce atransient increase in blood vessel permeability to macromolecules. Theterm “VEGF” or “VEGF-A” is used to refer to the 165-amino acid humanvascular endothelial cell growth factor and related 121-, 145-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by, e.g., Leung et al. Science, 246: 1306 (1989), and Houck etal. Mol. Endocrin., 5: 1806 (1991), together with the naturallyoccurring allelic and processed forms thereof. VEGF-A is part of a genefamily including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF.VEGF-A primarily binds to two high affinity receptor tyrosine kinases,VEGFR-1 (Fit-1) and VEGFR-2 (Flk-1 KDR), the latter being the majortransmitter of vascular endothelial cell mitogenic signals of VEGF-A.

“T-cell immunoreceptor with Ig and ITIM domains (TIGIT)”, is an immunereceptor found on T-cells and natural killer cells (NK cells), asdescribed by Yu X, et al., Nat Immunol. 10 (1): 48-57 (2009). It is alsoreferred to as WUCAM and Vstm3. TIGIT binds to CD155(PVR) with highaffinity on, for example, dendritic cells (DCs) and macrophages. TIGITalso binds to CD112(PVRL2), but with lower affinity. See, also,Anderson, A., et al., Immunity, 44(5):989-1004 (2016). The human TIGITsequence can be found on UniProtKB under accession number Q495A1.

“4-1BB” also referred to as CD137 or “Tumor necrosis factor receptorsuperfamily member 9 (TNFRSF9 or TNR9)” is a receptor that contributesto the clonal expansion, survival, and development of T cells. Thereceptor can be involved in inducing proliferation in peripheralmonocytes, enhancing T cell apoptosis induced by TCR/CD3 triggeredactivation, and regulating CD28 co-stimulation to promote Th1 cellresponses. The expression of this receptor is induced by lymphocyteactivation. TRAF adaptor proteins have been shown to bind to thisreceptor and transduce the signals leading to activation of NF-kappaB.See, for example, Zhou, Z., et al., Immunol. Lett., 45(1-2):67-73 (1995)and Alderson M. R., et al., Eur J Immunol., 24(9):2219-27 (1994). Thehuman 4-1BB (TNR) can be found on UniProtKB under accession numberQ07011.

“IFNα” or “IFN-α” also referred to as “interferon alpha” is a cytokinethat can stimulate the production of a protein kinase and anoligoadenylate synthetase (OAS). It is produced by immune cells such asmacrophages and has antiviral, antiproliferative, and immunomodulatoryproperties. IFNα binds to the interferon alpha receptor and activatesdownstream signaling via two cytoplasmic tyrosine kinases, Janus kinase1 (JAK1) and tyrosine kinase 2 (TYK2). The tyrosine kinases activate theJAK/STAT pathway to mediate antiviral and inflammatory effects of IFNα.See, e.g., Taniguchi et al., Nature, 285 (5766), 547-549 (1980) and Zoonet al., J. Biol. Chem., 267:15210-15216, (1992). The human IFNα sequencecan be found on UniProTKB under accession number P01562 or P01563

“GITR” also referred to as “glucocorticoid-induced TNFR-relatedprotein,” “tumor necrosis factor receptor superfamily member 18,”“TNFRSF18,” “activation-inducible TNFR family receptor,” and “AITR” is amember of the TNFR superfamily of receptors and is a co-stimulatoryimmune checkpoint molecule. GITR is a receptor that is involved ininhibiting the suppressive activity of T regulatory cells and extendingthe survival of T effector cells. GITR can be upregulated (induced) onactivated T cells. See, e.g., Shimizu et al., Nat. Immunol.,3(2):135-142 (2002) and Gurney et al., Curr. Biol., 9(4):215-218 (1999).The human GITR sequence can be found on UniProTKB under accession numberQ9Y5U5.

“OX40” also referred to as “CD134,” “tumor necrosis factor receptorsuperfamily, member 4,” “TNFRSF4,” and “OX40 receptor” is a member ofthe TNFR superfamily of receptors and is a secondary co-stimulatoryimmune checkpoint molecule. Expression of OX40 is dependent on theactivation of T-cells. Its ligand, OX40L binds to OX40 receptors onT-cells, thereby preventing the T-cells from dying and increasing theproduction of cytokines. It has been shown that OX40 plays a role in Th1and Th2-mediated immune responses. See, e.g., Arch and Thompson, Mol.Cell. Biol. 18 (1):558-65 (1998), Baum et al., Circ. Shock, 44:30-34(1994), and Latsa et al., Eur. J. Immunol., 24(3):677-683 (1994). Thehuman OX40 sequence can be found on UniProTKB under accession numberP43489.

“CD40” is a member of the TNFR superfamily of receptors and is acostimulatory protein found on immune cells such as antigen presentingcells (e.g., dendritic cells, B-cells, and macrophages). Its ligand,CD40L (CD154 or TNFSFS) is expressed on T helper cells and upon bindingto CD40, activates the antigen presenting cells. CD40 is also expressedby endothelial cells, smooth muscle cells, fibroblasts, epithetialcells, and tumor cells. See, e.g., Grewal and Flavell, Annual Review ofImmunology, 16:111-35 (1998) and Chatzigeorgiou et al., BioFactors(Oxford, England), 35(6):474-83 (2009). The human CD40 sequence can befound on UniProTKB under accession number P25942.

“ICOS” also referred to as “inducible T-cell costimulatory” or “CD278”is a member of the CD28 superfamily of costimulatory molecules and isexpressed on activated T cells. ICOS plays a role in regulating adaptiveimmune response, such as by enhancing T-cell proliferation and secretionof cytokines. See, e.g., Hutloff et al., Nature, 397(6716):263-6 (1999)and Beier et al., Eur. J. Immunol., 30:3707-3717 (2000). The human ICOSsequence can be found on UniProTKB under accession number Q9Y6W8.

“CD28” is a receptor for CD80 (B7.1) and CD86 (B7.2) which are expressedon antigen presenting cells. CD28 which is expressed on naïve T cells isinvolved in T cell activation and survival, and in the production ofcytokines. See, e.g., Linsley and Ledbetter, Annu. Rev. Immunol.11:191-212 (1993), Nunes et al., J. Biol. Chem. 271(3): 1591-8 (1996),and Bour-Jordan and Blueston, J. Clin. Immunol. 22(1):1-7 (2002). Thehuman CD28 sequence can be found on UniProTKB under accession numberP10747.

As used herein, “immune checkpoint” refers to stimulatory and inhibitorysignals that regulate the amplitude and quality of T cell receptorrecognition of an antigen. In certain embodiments, the immune checkpointis an inhibitory signal. In certain embodiments, the inhibitory signalis the interaction between PD-1 and PD-L1. In certain embodiments, theinhibitory signal is the interaction between CTLA-4 and CD80 or CD86 todisplace CD28 binding. In certain embodiments the inhibitory signal isthe interaction between LAG-3 and MHC class II molecules. In certainembodiments, the inhibitory signal is the interaction between TIM-3 andgalectin 9. In certain embodiments, the inhibitory signal is theinteraction between TIGIT and CD155. In certain embodiments, the immunecheckpoint is a stimulatory signal, which includes, for example, signalsthat reduce and/or eliminate immune suppression. In certain embodiments,the stimulatory signal is between 4-1BB/CD137 and its ligand (forexample, CD137L). In certain embodiments, the stimulatory signal isbetween IFNα and its ligand. In certain embodiments, the stimulatorysignal results from a modulation of the interaction between GITR and itsligand (for example, GITRL). In certain embodiments, the stimulatorysignal from a modulation of the interaction between OX40 and its ligand(for example, OX40L). In certain embodiments, the stimulatory signalfrom a modulation of the interaction between ICOS and its ligand. Incertain embodiments, the stimulatory signal from a modulation of theinteraction between IFNα and its receptor (for example, IFNαR). Incertain embodiments, the stimulatory signal from a modulation of theinteraction between CD28 and its ligand (for example, CD80 or CD86). Incertain embodiments, the stimulatory signal from a modulation of theinteraction between CD40 and its ligand (for example, CD40L).

As used herein, “immune checkpoint blocker” or “immune checkpointinhibitor” or “immune checkpoint modulator” refers to a molecule thatreduces, inhibits, interferes with or modulates one or more checkpointproteins or other proteins in the immune system pathways. In certainembodiments, the immune checkpoint inhibitor prevents inhibitory signalsassociated with the immune checkpoint. In certain embodiments, theimmune checkpoint inhibitor is an antibody, or fragment thereof, thatdisrupts inhibitory signaling associated with the immune checkpoint. Incertain embodiments, the immune checkpoint inhibitor is a small moleculethat disrupts inhibitory signaling. In certain embodiments, the immunecheckpoint inhibitor is an antibody, fragment thereof, or antibodymimic, that prevents the interaction between checkpoint blockerproteins, e.g., an antibody, or fragment thereof, that prevents theinteraction between PD-1 and PD-L1. In certain embodiments, the immunecheckpoint inhibitor is an antibody, or fragment thereof, that preventsthe interaction between CTLA-4 and CD80 or CD86. In certain embodiments,the immune checkpoint inhibitor is an antibody, or fragment thereof,that prevents the interaction between LAG-3 and MHC class II molecules.In certain embodiments the, the immune checkpoint inhibitor is anantibody, or fragment thereof, that prevents the interaction betweenTIM-3 and galectin9. The checkpoint blocker may also be in the form ofthe soluble form of the molecules (or mutation thereof) themselves,e.g., a soluble PD-L1 or PD-L1 fusion, as well as a soluble TIGIT orTIGIT fusion.

As used herein, “immune checkpoint enhancer” or “immune checkpointstimulator” or “immune checkpoint modulator” refers to a molecule thatenhances, increases, or modulates one or more checkpoint proteins orother proteins in the immune system pathways. In certain embodiments,the immune checkpoint stimulator induces inhibitory signals associatedwith the immune checkpoint. In certain embodiments, the immunecheckpoint stimulator reduces signals associated with immune checkpointsuppression. In certain embodiments, the immune checkpoint stimulator isan antibody, or fragment thereof, that increases inhibitory signalingassociated with the immune checkpoint. In certain embodiments, theimmune checkpoint stimulator is an antibody, or fragment thereof, thatreduces signaling associated with the immune checkpoint suppression. Incertain embodiments, the immune checkpoint stimulator is a smallmolecule that disrupts the suppression of inhibitory signaling. Incertain embodiments, the immune checkpoint stimulator is an antibody,fragment thereof, or antibody mimic, that prevents the interactionbetween checkpoint inhibitor proteins, e.g., an antibody, or fragmentthereof. In certain embodiments, the immune checkpoint stimulator is anantibody, or fragment thereof, that prevents the interaction between4-1BB (CD137), IFNα, GITR, and OX40 and their respective associatedbinding partner. The immune checkpoint stimulator may also be in theform of the soluble form of the molecules (or mutation thereof)themselves, e.g., a soluble PD-L1 or PD-L1 fusion, as well as a solubleTIGIT or TIGIT fusion.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., cancer, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FAST A, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al, J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly-ser polypeptide linker comprises the amino acid sequenceSer(Gly4Ser)n. In one embodiment, n=1. In one embodiment, n=2. Inanother embodiment, n=3, i.e., Ser(Gly4Ser)3. In another embodiment,n=4, i.e., Ser(Gly4Ser)4. In another embodiment, n=5. In yet anotherembodiment, n=6. In another embodiment, n=7. In yet another embodiment,n=8. In another embodiment, n=9. In yet another embodiment, n=10.Another exemplary gly-ser polypeptide linker comprises the amino acidsequence (Gly4Ser)n. In one embodiment, n=1. In one embodiment, n=2. Ina preferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6. Another exemplarygly-ser polypeptide linker comprises the amino acid sequence (Gly3Ser)n.In one embodiment, n=1. In one embodiment, n=2. In a preferredembodiment, n=3. In another embodiment, n=4. In another embodiment, n=5.In yet another embodiment, n=6.

As used herein, “half-life” refers to the time taken for the serum orplasma concentration of a polypeptide to reduce by 50%, in vivo, forexample due to degradation and/or clearance or sequestration by naturalmechanisms. The extended-PK IL-2 used herein is stabilized in vivo andits half-life increased by, e.g., fusion to HSA, MSA or Fc, throughPEGylation, or by binding to serum albumin molecules (e.g., human serumalbumin) which resist degradation and/or clearance or sequestration. Thehalf-life can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may for example generally involve thesteps of suitably administering a suitable dose of the amino acidsequence or compound of the invention to a subject; collecting bloodsamples or other samples from said subject at regular intervals;determining the level or concentration of the amino acid sequence orcompound of the invention in said blood sample; and calculating, from (aplot of) the data thus obtained, the time until the level orconcentration of the amino acid sequence or compound of the inventionhas been reduced by 50% compared to the initial level upon dosing.Further details are provided in, e.g., standard handbooks, such asKenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al., Pharmacokinetic Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

As used herein, a “small molecule” is a molecule with a molecular weightbelow about 500 Daltons.

As used herein, “therapeutic protein” refers to any polypeptide,protein, protein variant, fusion protein and/or fragment thereof whichmay be administered to a subject as a medicament. An exemplarytherapeutic protein is an interleukin, e.g., IL-7.

As used herein, “synergy” or “synergistic effect” with regard to aneffect produced by two or more individual components refers to aphenomenon in which the total effect produced by these components, whenutilized in combination, is greater than the sum of the individualeffects of each component acting alone.

The term “sufficient amount” or “amount sufficient to” means an amountsufficient to produce a desired effect, e.g., an amount sufficient toreduce the size of a tumor.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

As used herein, “combination therapy” embraces administration of eachagent or therapy in a sequential manner in a regiment that will providebeneficial effects of the combination and co-administration of theseagents or therapies in a substantially simultaneous manner. Combinationtherapy also includes combinations where individual elements may beadministered at different times and/or by different routes but which actin combination to provide a beneficial effect by co-action orpharmacokinetic and pharmacodynamics effect of each agent or tumortreatment approaches of the combination therapy.

As used herein, “about” will be understood by persons of ordinary skilland will vary to some extent depending on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill given the context in which it is used, “about” will meanup to plus or minus 10% of the particular value.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Various aspects described herein are described in further detail in thefollowing subsections.

III. IL-2 And Extended-PK IL-2

The integrin-binding polypeptide-Fc fusions of the invention can be usedin absence of IL-2 and/or extended IL-2. In some embodiments, theintegrin-binding polypeptide-Fc fusions of the invention can be used incombination with IL-2 and do not require the use of an extendedhalf-life IL-2. In some embodiments, integrin-binding polypeptide-Fcfusions can also be used in combination with half-life extended IL-2.

Interleukin-2 (IL-2) is a cytokine that induces proliferation ofantigen-activated T cells and stimulates natural killer (NK) cells. Thebiological activity of IL-2 is mediated through a multi-subunit IL-2receptor complex (IL-2R) of three polypeptide subunits that span thecell membrane: p55 (IL-2Ra, the alpha subunit, also known as CD25 inhumans), p75 (IL-2RP, the beta subunit, also known as CD 122 in humans)and p64 (IL-2Ry, the gamma subunit, also known as CD 132 in humans). Tcell response to IL-2 depends on a variety of factors, including: (1)the concentration of IL-2; (2) the number of IL-2R molecules on the cellsurface; and (3) the number of IL-2R occupied by IL-2 (i.e., theaffinity of the binding interaction between IL-2 and IL-2R (Smith, “CellGrowth Signal Transduction is Quantal” In Receptor Activation byAntigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)).The IL-2TL-2R complex is internalized upon ligand binding and thedifferent components undergo differential sorting. IL-2Ra is recycled tothe cell surface, while IL-2 associated with the IL-2TL-2Rpy complex isrouted to the lysosome and degraded. When administered as an intravenous(i.v.) bolus, IL-2 has a rapid systemic clearance (an initial clearancephase with a half-life of 12.9 minutes followed by a slower clearancephase with a half-life of 85 minutes) (Konrad et al., Cancer Res.50:2009-2017, 1990).

Thus, in some embodiments, IL-2 therapy, such as systemic IL-2, isadministered to a subject in an effective amount in combination with anintegrin-binding-Fc fusion protein, and optionally an immune checkpointinhibitor.

However, outcomes of systemic IL-2 administration in cancer patients arefar from ideal. While 15 to 20 percent of patients respond objectivelyto high-dose IL-2, the great majority do not, and many suffer severe,life-threatening side effects, including nausea, confusion, hypotension,and septic shock. The severe toxicity associated with IL-2 treatment islargely attributable to the activity of natural killer (NK) cells. NKcells express the intermediate-affinity receptor, IL-2Rβy_(c), and thusare stimulated at nanomolar concentrations of IL-2, which do in factresult in patient sera during high-dose IL-2 therapy. Attempts to reduceserum concentration, and hence selectively stimulateIL-2Raβy_(c)-bearing cells, by reducing dose and adjusting dosingregimen have been attempted, and while less toxic, such treatments werealso less efficacious. Given the toxicity issues associated with highdose IL-2 cancer therapy, numerous groups have attempted to improveanti-cancer efficacy of IL-2 by simultaneously administering therapeuticantibodies. Yet, such efforts have been largely unsuccessful, yieldingno additional or limited clinical benefit compared to IL-2 therapyalone. Accordingly, novel IL-2 therapies are needed to more effectivelycombat various cancers.

Applicants recently discovered that the ability of IL-2 to controltumors in various cancer models could be substantially increased byattaching IL-2 to a pharmacokinetic modifying group. The resultingmolecule, hereafter referred to as “extended-pharmacokinetic (PK) IL-2,”has a prolonged circulation half-life relative to free IL-2. Theprolonged circulation half-life of extended-PK IL-2 permits in vivoserum IL-2 concentrations to be maintained within a therapeutic range,leading to the enhanced activation of many types of immune cells,including T cells. Because of its favorable pharmacokinetic profile,extended-PK IL-2 can be dosed less frequently and for longer periods oftime when compared with unmodified IL-2. Extended-PK IL-2 is describedin detail in International Patent Application NO. PCT/US2013/042057,filed May 21, 2013, and claiming the benefit of priority to U.S.Provisional Patent Application No. 61/650,277, filed May 22, 2012. Theentire contents of the foregoing applications are incorporated byreference herein.

1. IL-2 AND MUTANTS THEREOF

In certain embodiments, an effective amount of human IL-2 isadministered systemically. In some embodiments, an effective amount ofan extended-PK IL-2 is administered systemically. In one embodiment, theIL-2 is a human recombinant IL-2 such as Proleukin® (aldesleukin).Proleukin® is a human recombinant interleukin-2 product produced in E.coli. Proleukin® differs from the native interleukin-2 in the followingways: a) it is not glycosylated; b) it has no N-terminal alanine; and c)it has serine substituted for cysteine at amino acid positions 125.Proleukin® exists as biologically active, non-covalently boundmicroaggregates with an average size of 27 recombinant interleukin-2molecules. Proleukin® (aldesleukin) is administered by intravenousinfusion. In some aspects, the IL-2 portion of the extended-PK IL-2 iswild-type IL-2 (e.g., human IL-2 in its precursor form (SEQ ID NO: 33from International Patent Publication WO 2016/025642, incorporatedherein by reference in its entirety) or mature IL-2 (SEQ ID NO: 35 fromInternational Patent Publication WO 2016/025642, incorporated herein byreference in its entirety)).

In certain embodiments, the extended-PK IL-2 is mutated such that it hasan altered affinity (e.g., a higher affinity) for the IL-2R alphareceptor compared with unmodified IL-2.

Site-directed mutagenesis can be used to isolate IL-2 mutants thatexhibit high affinity binding to CD25, i.e., IL-2Ra, as compared towild-type IL-2. Increasing the affinity of IL-2 for IL-2Ra at the cellsurface will increase receptor occupancy within a limited range of IL-2concentration, as well as raise the local concentration of IL-2 at thecell surface.

In certain embodiments, the invention features IL-2 mutants, which maybe, but are not necessarily, substantially purified and which canfunction as high affinity CD25 binders. IL-2 is a T cell growth factorthat induces proliferation of antigen-activated T cells and stimulationof NK cells. Exemplary IL-2 mutants which are high affinity bindersinclude those described in WO 2013/177187A2 (herein incorporated byreference in its entirety), such as those with amino acid sequences setforth in SEQ ID Nos: 7, 23, 25, 27, 29, and 31. Further exemplary IL-2mutants with increased affinity for CD25 are disclosed in U.S. Pat. No.7,569,215, the contents of which are incorporated herein by reference.In one embodiment, the IL-2 mutant does not bind to CD25, e.g., thosewith amino acid sequences set forth in SEQ ID Nos: 9 and 11.

IL-2 mutants include an amino acid sequence that is at least 80%identical to SEQ ID NO: 33 (from International Patent Publication WO2016/025642, incorporated herein by reference in its entirety) that bindCD25. For example, an IL-2 mutant can have at least one mutation (e.g.,a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues)that increases the affinity for the alpha subunit of the IL-2 receptorrelative to wild-type IL-2. It should be understood that mutationsidentified in mouse IL-2 may be made at corresponding residues in fulllength human IL-2 (nucleic acid sequence (accession: NM000586) of SEQ IDNO: 32 (from International Patent Publication WO 2016/025642,incorporated herein by reference in its entirety); amino acid sequence(accession: P60568 of SEQ ID NO: 33 from International PatentPublication WO 2016/025642, incorporated herein by reference in itsentirety) or human IL-2 without the signal peptide (nucleic acidsequence of SEQ ID NO: 34 (from International Patent Publication WO2016/025642, incorporated herein by reference in its entirety); aminoacid sequence of SEQ ID NO: 35 (from International Patent Publication WO2016/025642, incorporated herein by reference in its entirety).Accordingly, in certain embodiments, the IL-2 moiety of the extended-PKIL-2 is human IL-2. In other embodiments, the IL-2 moiety of theextended-PK IL-2 is a mutant human IL-2.

IL-2 mutants can be at least or about 50%, at least or about 65%, atleast or about 70%, at least or about 80%, at least or about 85%, atleast or about 87%, at least or about 90%, at least or about 95%, atleast or about 97%, at least or about 98%, or at least or about 99%identical in amino acid sequence to wild-type IL-2 (in its precursorform or, preferably, the mature form). The mutation can consist of achange in the number or content of amino acid residues. For example, theIL-2 mutants can have a greater or a lesser number of amino acidresidues than wild-type IL-2. Alternatively, or in addition, IL-2mutants can contain a substitution of one or more amino acid residuesthat are present in the wild-type IL-2.

By way of illustration, a polypeptide that includes an amino acidsequence that is at least 95% identical to a reference amino acidsequence of SEQ ID NO: 33 is a polypeptide that includes a sequence thatis identical to the reference sequence except for the inclusion of up tofive alterations of the reference amino acid of SEQ ID NO: 33 (fromInternational Patent Publication WO 2016/025642, incorporated herein byreference in its entirety). For example, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino (N—) or carboxy (C—) terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

The substituted amino acid residue(s) can be, but are not necessarily,conservative substitutions, which typically include substitutions withinthe following groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. These mutations can be atamino acid residues that contact IL-2Ra.

In general, the polypeptides used in the practice of the instantinvention will be synthetic, or produced by expression of a recombinantnucleic acid molecule. In the event the polypeptide is an extended-PKIL-2 (e.g., a fusion protein containing at least IL-2 and a heterologouspolypeptide, such as a hexa-histidine tag or hemagglutinin tag or an Fcregion or human serum albumin), it can be encoded by a hybrid nucleicacid molecule containing one sequence that encodes IL-2 and a secondsequence that encodes all or part of the heterologous polypeptide.

The techniques that are required to make IL-2 mutants are routine in theart, and can be performed without resort to undue experimentation by oneof ordinary skill in the art. For example, a mutation that consists of asubstitution of one or more of the amino acid residues in IL-2 can becreated using a PCR-assisted mutagenesis technique (e.g., as known inthe art and/or described herein for the creation of IL-2 mutants).Mutations that consist of deletions or additions of amino acid residuesto an IL-2 polypeptide can also be made with standard recombinanttechniques. In the event of a deletion or addition, the nucleic acidmolecule encoding IL-2 is simply digested with an appropriaterestriction endonuclease. The resulting fragment can either be expresseddirectly or manipulated further by, for example, ligating it to a secondfragment. The ligation may be facilitated if the two ends of the nucleicacid molecules contain complementary nucleotides that overlap oneanother, but blunt-ended fragments can also be ligated. PCR-generatednucleic acids can also be used to generate various mutant sequences.

In addition to generating IL-2 mutants via expression of nucleic acidmolecules that have been altered by recombinant molecular biologicaltechniques, IL-2 mutants can be chemically synthesized. Chemicallysynthesized polypeptides are routinely generated by those of skill inthe art.

As noted above, IL-2 can also be prepared as fusion or chimericpolypeptides that include IL-2 and a heterologous polypeptide (i.e., apolypeptide that is not IL-2). The heterologous polypeptide can increasethe circulating half-life of the chimeric polypeptide in vivo, and may,therefore, further enhance the properties of IL-2. As discussed infurther detail infra, the polypeptide that increases the circulatinghalf-life may be serum albumin, such as human or mouse serum albumin.

In other embodiments, the chimeric polypeptide can include IL-2 and apolypeptide that functions as an antigenic tag, such as a FLAG sequence.FLAG sequences are recognized by biotinylated, highly specific,anti-FLAG antibodies, as described herein (see also Blanar et al,Science 256: 1014, 1992; LeClair et al, Proc. Natl. Acad. Sci. USA89:8145, 1992). In certain embodiments, the chimeric polypeptide furthercomprises a C-terminal c-myc epitope tag.

Chimeric polypeptides can be constructed using no more than conventionalmolecular biological techniques, which are well within the ability ofthose of ordinary skill in the art to perform.

2. Nucleic Acid Molecules Encoding IL-2

IL-2, either alone or as a part of a chimeric polypeptide, such as thosedescribed herein, can be obtained by expression of a nucleic acidmolecule. Thus, nucleic acid molecules encoding polypeptides containingIL-2 or an IL-2 mutant are considered within the scope of the invention,such as those with nucleic acid sequences set forth in SEQ ID Nos: 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. Just as IL-2mutants can be described in terms of their identity with wild-type IL-2,the nucleic acid molecules encoding them will necessarily have a certainidentity with those that encode wild-type IL-2. For example, the nucleicacid molecule encoding an IL-2 mutant can be at least 50%, at least 65%,preferably at least 75%, more preferably at least 85%, and mostpreferably at least 95% (e.g., 99%) identical to the nucleic acidencoding full length wild-type IL-2 (e.g., SEQ ID NO: 32 fromInternational Patent Publication WO 2016/025642, incorporated herein byreference in its entirety) or wild-type IL-2 without the signal peptide(e.g., SEQ ID NO: 34 from International Patent Publication WO2016/025642, incorporated herein by reference in its entirety).

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide. These nucleic acid molecules can consist of RNA or DNA(for example, genomic DNA, cDNA, or synthetic DNA, such as that producedby phosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

The nucleic acid molecules are not limited to sequences that encodepolypeptides; some or all of the non-coding sequences that lie upstreamor downstream from a coding sequence (e.g., the coding sequence of IL-2)can also be included. Those of ordinary skill in the art of molecularbiology are familiar with routine procedures for isolating nucleic acidmolecules. They can, for example, be generated by treatment of genomicDNA with restriction endonucleases, or by performance of the polymerasechain reaction (PCR). In the event the nucleic acid molecule is aribonucleic acid (RNA), molecules can be produced, for example, by invitro transcription.

The isolated nucleic acid molecules can include fragments not found assuch in the natural state. Thus, the invention encompasses use ofrecombinant molecules, such as those in which a nucleic acid sequence(for example, a sequence encoding an IL-2 mutant) is incorporated into avector (e.g., a plasmid or viral vector) or into the genome of aheterologous cell (or the genome of a homologous cell, at a positionother than the natural chromosomal location).

As described above, IL-2 mutants of the invention may exist as a part ofa chimeric polypeptide. In addition to, or in place of, the heterologouspolypeptides described above, a nucleic acid molecule of the inventioncan contain sequences encoding a “marker” or “reporter.” Examples ofmarker or reporter genes include β-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo^(r), G418^(r)), dihydrofolate reductase (DHFR),hygromycin-B-hosphotransferase (HPH), thymidine kinase (TK), lacz(encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures associatedwith the practice of the invention, skilled artisans will be aware ofadditional useful reagents, for example, of additional sequences thatcan serve the function of a marker or reporter.

The nucleic acid molecules of the invention can be obtained byintroducing a mutation into IL-2-encoding DNA obtained from anybiological cell, such as the cell of a mammal. Thus, the nucleic acidsused herein (and the polypeptides they encode) can be those of a mouse,rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, orcat. Typically, the nucleic acid molecules will be those of a human.

3. Extended-PK Groups

As described supra, IL-2 or mutant IL-2 is fused to an extended-PKgroup, which increases circulation half-life. Non-limiting examples ofextended-PK groups are described infra. It should be understood thatother PK groups that increase the circulation half-life of IL-2, orvariants thereof, are also applicable to extended-PK IL-2.

In certain embodiments, the serum half-life of extended-PK IL-2 isincreased relative to IL-2 alone (i.e., IL-2 not fused to an extended-PKgroup). In certain embodiments, the serum half-life of extended-PK IL-2is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or1000% longer relative to the serum half-life of IL-2 alone. In otherembodiments, the serum half-life of the extended-PK IL-2 is at least1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold,6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold,20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or50-fold greater than the serum half-life of IL-2 alone. In certainembodiments, the serum half-life of the extended-PK IL-2 is at least 10hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200hours.

4. Serum Albumin and Serum Albumin Binding Proteins

In certain embodiments, the extended-PK group is a serum albumin, or

fragments thereof. Methods of fusing serum albumin to proteins aredisclosed in, e.g., US2010/0144599, US2007/0048282, and US2011/0020345,which are herein incorporated by reference in their entirety. In certainembodiments, the extended-PK group is HSA, or variants or fragmentsthereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO2011/124718, WO 2013/075066, and WO 2011/0514789.

In certain embodiments, the extended-PK group is a serum albumin bindingprotein such as those described in US2005/0287153, US2007/0003549,US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, andWO2009/133208, which are herein incorporated by reference in theirentirety.

1. Pegylation

In certain embodiments, an extended-PK IL-2 used herein includes apolyethylene glycol (PEG) domain. PEGylation is well known in the art toconfer increased circulation half-life to proteins. Methods ofPEGylation are well known and disclosed in, e.g., U.S. Pat. Nos.7,610,156, 7,847,062, all of which are hereby incorporated by reference.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X-0(CH2CH20)_(n)-iCH2CH₂OH,where n is 20 to 2300 and X is H or a terminal modification, e.g., aC1.4 alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH3 (“mefhoxy PEG”).PEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat.No. 5,932,462, both of which are hereby incorporated by reference. Oneform of PEGs includes two PEG side-chains (PEG2) linked via the primaryamino groups of a lysine (Monfardini et al., Bioconjugate Chem 1995;6:62-9).

In one embodiment, pegylated IL-2 is produced by site-directedpegylation, particularly by conjugation of PEG to a cysteine moiety atthe N- or C-terminus. A PEG moiety may also be attached by otherchemistry, including by conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al, JBC 1977; 252:3571 and JBC 1977; 252:3582, and Harriset. al, in: Polyethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22).

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to IL-2. The number of repeating units “n” in the PEG isapproximated for the molecular mass described in Daltons. It ispreferred that the combined molecular mass of PEG on an activated linkeris suitable for pharmaceutical use. Thus, in one embodiment, themolecular mass of the PEG molecules does not exceed 100,000 Da. Forexample, if three PEG molecules are attached to a linker, where each PEGmolecule has the same molecular mass of 12,000 Da (each n is about 270),then the total molecular mass of PEG on the linker is about 36,000 Da(total n is about 820). The molecular masses of the PEG attached to thelinker can also be different, e.g., of three molecules on a linker twoPEG molecules can be 5,000 Da each (each n is about 110) and one PEGmolecule can be 12,000 Da (n is about 270).

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated IL-2 will be used therapeutically, thedesired dosage, circulation time, resistance to proteolysis,immunogenicity, and other considerations. For a discussion of PEG andits use to enhance the properties of proteins, see N. V. Katre, AdvancedDrug Delivery Reviews 1993; 10:91-114.

In one embodiment of the invention, PEG molecules may be activated toreact with amino groups on IL-2 such as with lysines (Bencham C. O. etal., Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al, Appl.Biochem., 11, 141 (1985); Zalipsky, S. et al, Polymeric Drugs and DrugDelivery Systems, adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky,S. et al, Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al,Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).

In one embodiment, carbonate esters of PEG are used to form the PEG-IL-2conjugates. N,N′-disuccinimidylcarbonate (DSC) may be used in thereaction with PEG to form active mixed PEG-succinimidyl carbonate thatmay be subsequently reacted with a nucleophilic group of a linker or anamino group of IL-2 (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In asimilar type of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of IL-2 can be performed according to the methods of thestate of the art, for example by reaction of IL-2 with electrophilicallyactive PEGs (Shearwater Corp., USA, www.shearwatercorp.com). PreferredPEG reagents are, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA),butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C, et al.,Bioconjugate Chem. 6 (1995) 62-69).

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson IL-2 (Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991);Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson et al,Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos.6,610,281 and 5,766,897 describe exemplary reactive PEG species that maybe coupled to sulfhydryl groups.

In certain embodiments where PEG molecules are conjugated to cysteineresidues on IL-2 the cysteine residues are native to IL-2 whereas inother embodiments, one or more cysteine residues are engineered intoIL-2. Mutations may be introduced into the coding sequence of IL-2 togenerate cysteine residues. This might be achieved, for example, bymutating one or more amino acid residues to cysteine. Preferred aminoacids for mutating to a cysteine residue include serine, threonine,alanine and other hydrophilic residues. Preferably, the residue to bemutated to cysteine is a surface-exposed residue. Algorithms arewell-known in the art for predicting surface accessibility of residuesbased on primary sequence or a protein.

In another embodiment, pegylated IL-2 comprises one or more PEGmolecules covalently attached to a linker.

In some embodiments, the pegylated IL-2 is NKTR-214. NKTR-12 is an IL-2conjugated to 6 releasable polyethylene glycol PEG chains. In vivo, thePEG chains slowly release to generate active IL-2 conjugates, asdescribed in Charych, D. H., et al., Clinical Cancer Res.; 22(3):680-690 (2016).

In one embodiment, IL-2 is pegylated at the C-terminus. In a specificembodiment, a protein is pegylated at the C-terminus by the introductionof C-terminal azido-methionine and the subsequent conjugation of amethyl-PEG-triarylphosphine compound via the Staudinger reaction. ThisC-terminal conjugation method is described in Cazalis et al., C-TerminalSite-Specific PEGylation of a Truncated Thrombomodulin Mutant withRetention of Full Bioactivity, Bioconjug Chem. 2004; 15(5): 1005-1009.

Monopegylation of IL-2 can also be achieved according to the generalmethods described in WO 94/01451. WO 94/01451 describes a method forpreparing a recombinant polypeptide with a modified terminal amino acidalpha-carbon reactive group. The steps of the method involve forming therecombinant polypeptide and protecting it with one or more biologicallyadded protecting groups at the N-terminal alpha-amine and C-terminalalpha-carboxyl. The polypeptide can then be reacted with chemicalprotecting agents to selectively protect reactive side chain groups andthereby prevent side chain groups from being modified. The polypeptideis then cleaved with a cleavage reagent specific for the biologicalprotecting group to form an unprotected terminal amino acid alpha-carbonreactive group. The unprotected terminal amino acid alpha-carbonreactive group is modified with a chemical modifying agent. The sidechain protected terminally modified single copy polypeptide is thendeprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of IL-2 to activated PEG in the conjugation reaction can befrom about 1:0.5 to 1:50, between from about 1:1 to 1:30, or from about1:5 to 1:15. Various aqueous buffers can be used to catalyze thecovalent addition of PEG to IL-2, or variants thereof. In oneembodiment, the pH of a buffer used is from about 7.0 to 9.0. In anotherembodiment, the pH is in a slightly basic range, e.g., from about 7.5 to8.5. Buffers having a pKa close to neutral pH range may be used, e.g.,phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated IL-2, such as size exclusion (e.g. gelfiltration) and ion exchange chromatography. Products may also beseparated using SDS-PAGE. Products that may be separated include mono-,di-, tri-poly- and un-pegylated IL-2 as well as free PEG. The percentageof mono-PEG conjugates can be controlled by pooling broader fractionsaround the elution peak to increase the percentage of mono-PEG in thecomposition.

In one embodiment, PEGylated IL-2 of the invention contains one, two ormore PEG moieties. In one embodiment, the PEG moiety(ies) are bound toan amino acid residue which is on the surface of the protein and/or awayfrom the surface that contacts CD25. In one embodiment, the combined ortotal molecular mass of PEG in PEG-IL-2 is from about 3,000 Da to 60,000Da, optionally from about 10,000 Da to 36,000 Da. In one embodiment, PEGin pegylated IL-2 is a substantially linear, straight-chain PEG.

In one embodiment, PEGylated IL-2 of the invention will preferablyretain at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of thebiological activity associated with the unmodified protein. In oneembodiment, biological activity refers to the ability to bind CD25. Theserum clearance rate of PEG-modified IL-2 may be decreased by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative to theclearance rate of the unmodified IL-2. PEG-modified IL-2 may have acirculation half-life which is enhanced relative to the half-life ofunmodified IL-2. The half-life of PEG-IL-2, or variants thereof, may beenhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by 1000%relative to the half-life of unmodified IL-2. In certain embodiments,the protein half-life is determined in vitro, such as in a bufferedsaline solution or in serum. In other embodiments, the protein half-lifeis an in vivo circulation half-life, such as the half-life of theprotein in the serum or other bodily fluid of an animal.

IV. Other Extended-PK Groups

In certain embodiments, the extended-PK group is transferrin, asdisclosed in U.S. Pat. Nos. 7,176,278 and 8,158,579, which are hereinincorporated by reference in their entirety.

In certain embodiments, the extended-PK group is a serum immunoglobulinbinding protein such as those disclosed in US2007/0178082, which isherein incorporated by reference in its entirety.

In certain embodiments, the extended-PK group is a fibronectin(Fn)-based scaffold domain protein that binds to serum albumin, such asthose disclosed in US2012/0094909, which is herein incorporated byreference in its entirety. Methods of making fibronectin-based scaffolddomain proteins are also disclosed in US2012/0094909. A non-limitingexample of a Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3protein that binds to human serum albumin.

V. Fe Domains

In certain embodiments, an extended-PK IL-2 includes an Fc domain, asdescribed in International Patent Publication No. WO 2013/177187. The Fcdomain does not contain a variable region that binds to antigen. Fcdomains useful for producing the extended-PK IL-2 described herein maybe obtained from a number of different sources. In certain embodiments,an Fc domain of the extended-PK IL-2 is derived from a humanimmunoglobulin. In a certain embodiment, the Fc domain is from a humanIgG1 constant region (for example, SEQ ID NO:126). The Fc domain ofhuman IgG1 is set forth in SEQ ID NO: 126. In certain embodiments, theFc domain of human IgG1 does not have the upper hinge region (SEQ ID NO:3 from International Patent Publication WO 2016/025642, incorporatedherein by reference in its entirety). It is understood, however, thatthe Fc domain may be derived from an immunoglobulin of another mammalianspecies, including for example, a rodent (e.g. a mouse, rat, rabbit,guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.Moreover, the Fc domain or portion thereof may be derived from anyimmunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and anyimmunoglobulin isotype, including IgG1 (for example, SEQ ID NO:126),IgG2 for example, SEQ ID NO:127, IgG3 (for example, SEQ ID NO:128), andIgG4 (for example, SEQ ID NO:129).

In some aspects, an extended-PK IL-2 includes a mutant Fc domain. Insome aspects, an extended-PK IL-2 includes a mutant, IgG1 Fc domain. Insome aspects, a mutant Fc domain comprises one or more mutations in thehinge, CH₂, and/or CH₃ domains. In some aspects, a mutant Fc domainincludes a D265A mutation.

In one embodiment, the extended-PK IL-2 of the invention lacks one ormore constant region domains of a complete Fc region, i.e., they arepartially or entirely deleted. In certain embodiments, the extended-PKIL-2 of the invention will lack an entire CH₂ domain. In certainembodiments, the extended-PK IL-2 of the invention comprise CH₂domain-deleted Fc regions derived from a vector (e.g., from IDECPharmaceuticals, San Diego) encoding an IgG1 human constant regiondomain (see, e.g., WO 02/060955A2 and WO 02/096948A2).

This exemplary vector is engineered to delete the CH₂ domain and providea synthetic vector expressing a domain-deleted IgG1 constant region. Itwill be noted that these exemplary constructs are preferably engineeredto fuse a binding CH₃ domain directly to a hinge region of therespective Fc domain.

VI. IFNα

In some embodiments, the IL-2 can be replaced with interferon-α (INFα).In some embodiments, the INFα is naturally occurring human INFα. In someembodiments, the INFα is a long acting INFα, such as those described inUS Patent Publication US 2006/0051859 with are fused with human serumalbumin. In some embodiments, the IFNα has the sequence from UniProtKBreference P01562 or P01563. In some embodiments, the IFNα is afunctional variant of the sequence from UniProtKB reference P01562 orP01563. In some embodiments, the IFNα comprises a sequence containing80%, 85%, 90%, 95%, or 100% identity to the sequence from UniProtKBreference P01562 or P01563 (SEQ ID NO:148 or 154). In some embodiments,the IFNα is Intron-A, commercially available from Merck (see, forexample, U.S. Pat. No. 6,610,830 andhttps://www.merck.com/product/usa/pi_circulars/i/intron_a/intron_a_pi.pdf).In some embodiments, the IFNα is PEG-IFNα. In some embodiments, the IFNαis Pegintron (see, for example, U.S. Pat. Nos. 6,610,830 and 6,180,096).In some embodiments, the IFNα is SYLATRON (see, for example, U.S. Pat.Nos. 6,610,830 and 6,180,096).

VII. Integrin and Knottin Polypeptides and Fe-Fusions

Integrins are a family of extracellular matrix adhesion receptors thatregulate a diverse array of cellular functions crucial to theinitiation, progression and metastasis of solid tumors. The importanceof integrins in tumor progression has made them an appealing target forcancer therapy and allows for the treatment of a variety of cancertypes. The integrins present on cancerous cells include α_(v)β₁,α_(v)β₃, α_(v)β₅, α_(v)β₆, and α₅β₁.

Knottin proteins are small compact peptides that have high thermal andproteolytic stability and are tolerant to mutagenesis, making them goodmolecular scaffolds. These peptides contain at least 3 disulfide bondsthat form a “knot” core. They also contain several loops exposed to thesurface, allowing these loops to bind targets. These loops can beengineered to bind specific targets with high affinity, making them auseful tool for therapy.

The present invention involves the use of a knottin polypeptide scaffoldengineered with an RGD sequence capable of binding integrins, fused toan Fc donor, which confers a therapeutic benefit (also referred to as“knottin-Fc”), herein collectively referred to as an integrin-bindingpolypeptide-Fc fusion. As described supra, Fc fragments have been addedto proteins and/or therapeutics to extend half-life. In the context ofintegrin-binding polypeptide-Fc fusion as used herein, the effectorfunction of Fc contributes to the treatment of a variety of cancers. Insome embodiments, this effect can find further use and/or be enhancedwhen used in conjunction with IL-2, including Proleukin and/orextended-PK IL-2. In some embodiments, an integrin-bindingpolypeptide-Fc fusion (also sometimes referred to as a knottin-Fc) thatbinds three integrins simultaneously, is used for example, anintegrin-binding polypeptide-Fc fusion that is selected from the groupconsisting of NOD201 (SEQ ID NO:139), NOD203 (SEQ ID NO:142), and NOD204(SEQ ID NO:143). In some embodiments, the integrin-bindingpolypeptide-Fc fusion is NOD201 (SEQ ID NO:139). In some embodiments,the integrin-binding polypeptide-Fc fusion is NOD203 (SEQ ID NO:142). Insome embodiments, the integrin-binding polypeptide-Fc fusion is NOD204(SEQ ID NO:143). In some embodiments, the integrin-bindingpolypeptide-Fc fusion comprises GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG, 2.5F,SEQ ID NO:130; GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG, 2.5FmodK, SEQ IDNO:131); GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132);GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133);GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134); orGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),operatively linked to an Fc domain. In some embodiments, theintegrin-binding polypeptide-Fc fusion comprisesGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG, 2.5F, SEQ ID NO:130;GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG, 2.5FmodK, SEQ ID NO:131;GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132);GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134); orGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135)operatively linked to an Fc domain, wherein said Fc domains is fromIgG1, IgG2, IgG3, and IgG4, including mouse or human. Exemplary IgGsequences are known in the art and can be found in FIG. 1 and Table 1above.

In some embodiments, the integrin-binding polypeptide-Fc fusions bind toone more integrins selected from α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, andα₅β₁ with high affinity. In some embodiments, the integrin-bindingpolypeptide-Fc fusions bind to two integrins selected from α_(v)β₁,α_(v)β₃, α_(v)β₅, α_(v)β₆, and α₅β₁ with high affinity. In someembodiments, the integrin-binding polypeptide-Fc fusions bind to threeintegrins selected from α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, and α₅β₁with high affinity. In some embodiments, the binding affinity is lessthan about 100 nM, less than about 50 nM, less than about 40 nM, lessthan about 30 nM, less than about 20 nM, less thank about 20 nM, lessthan about 10 nM, less than about 5 nM, less than about 4 nM, less thanabout 3 nM, less than about 2 nM, or less than about 1 nM. In someembodiments, the binding affinity is less than 5 nM. In someembodiments, the binding affinity is less than about 4 nM. In someembodiments, the binding affinity is less than about 3 nM. In someembodiments, the binding affinity is less than about 2 nM. In someembodiments, the binding affinity is less than about 1 nM. In someembodiments, the binding affinity is about 1.6 nM. In some embodiments,the binding affinity is about 1.5 nM. In some embodiments, the bindingaffinity is about 1 nM. In some embodiments, the binding affinity isabout 0.7 nM.

In some embodiments, NOD201 is highly stable to serum and thermalchallenge. In some embodiments, this stability is driven by Fc domainand not disulfide-bonded peptide. In some embodiments, no aggregation ordegradation of NOD201 occurs following extended incubation at 40° C. or5X freeze-thaw cycles

In silico immunogenicity analyses of NOD201 peptide (Antitope) has beenperformed, and iTope™ and TCED™ analyses were applied to the sequence inorder to identify peptides that were predicted to bind to human MHCclass II and/or share homology to known T cell epitopes. In thisanalysis, no matches to known T cell epitopes in the TCED™ wereidentified. In some embodiments, NOD201 does not contain non-germlinepromiscuous MHC Class II binding peptides. In some embodiments, the riskof NOD201 immunogenicity is therefore low. In some embodiments,immunogenicity of NOD201 is low.

1. FC Domains

The Fc domain does not contain a variable region that binds to antigen.Fc domains useful for the integrin-binding polypeptide-Fc fusionsdescribed herein may be obtained from a number of different sources. Incertain embodiments, an Fc domain of the extended-PK IL-2 is derivedfrom a human immunoglobulin. In a certain embodiment, the Fc domain isfrom a human IgG1 constant region (FIG. 1; SEQ ID NO:126). An exemplaryFc domain of human IgG1 is set forth in SEQ ID NO: 126 (FIG. 1). Incertain embodiments, the Fc domain of human IgG1 does not have the upperhinge region (FIG. 1 and Table 1). It is understood, however, that theFc domain may be derived from an immunoglobulin of another mammalianspecies, including for example, a rodent (e.g. a mouse, rat, rabbit,guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.Moreover, the Fc domain or portion thereof can be derived from anyimmunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and anyimmunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. The Fcdomain can be mouse or human.

In some embodiments, the integrin-binding polypeptide-Fc fusion includesa mutant Fc domain. In some embodiments, the integrin-bindingpolypeptide-Fc fusion includes a mutant, IgG1 Fc domain. In someembodiments, a mutant Fc domain comprises one or more mutations in thehinge, CH₂, and/or CH₃ domains. In some embodiments, a mutant Fc domainincludes a D265A mutation.

In some embodiments, the integrin-binding polypeptide-Fc fusion of theinvention lacks one or more constant region domains of a complete Fcregion, i.e., they are partially or entirely deleted. In certainembodiments, the integrin-binding polypeptide-Fc fusion of the inventionwill lack an entire CH₂ domain. In some embodiments, theintegrin-binding polypeptide-Fc fusion of the invention comprise CH₂domain-deleted Fc regions derived from a vector (e.g., from IDECPharmaceuticals, San Diego) encoding an IgG1 human constant regiondomain (see, e.g., WO 02/060955A2 and WO 02/096948A2).

In some embodiments, an exemplary vector is engineered to delete the CH₂domain and provide a synthetic vector expressing a domain-deleted IgG1constant region. It will be noted that these exemplary constructs arepreferably engineered to fuse a binding CH₃ domain directly to a hingeregion of the respective Fc domain.

2. Methods of Engineering Knottin Polypeptide Scaffolds

Knottin polypeptide scaffolds are used to insert an integrin-bindingsequence, preferably in the form of a loop, to confer specific integrinbinding. Integrin-binding is preferably engineered into a knottinpolypeptide scaffold by inserting an integrin-binding peptide sequence,such as an RGD peptide. In some embodiments, insertion of anintegrin-binding peptide sequence results in replacement of portion ofthe native knottin protein. For example, in one embodiment an RGDpeptide sequence is inserted into a native solvent exposed loop byreplacing all or a portion of the loop with an RGD-containing peptidesequence (e.g., 5-12 amino acid sequence) that has been selected forbinding to one or more integrins. The solvent-exposed loop (i.e., on thesurface) will generally be anchored by disulfide-linked cysteineresidues in the native knottin protein sequence. The integrin-bindingreplacement amino acid sequence can be obtained by randomizing codons inthe loop portion, expressing the engineered peptide, and selecting themutants with the highest binding to the predetermined ligand. Thisselection step may be repeated several times, taking the tightestbinding proteins from the previous step and re-randomizing the loops.

Integrin-binding polypeptides may be modified in a number of ways. Forexample, the polypeptide may be further cross-linked internally, or maybe cross-linked to each other, or the RGD loops may be grafted ontoother cross linked molecular scaffolds. There are a number ofcommercially available crosslinking reagents for preparing protein orpeptide bioconjugates. Many of these crosslinkers allow dimeric homo- orheteroconjugation of biological molecules through free amine orsulfhydryl groups in protein side chains. More recently, othercrosslinking methods involving coupling through carbohydrate groups withhydrazide moieties have been developed. These reagents have offeredconvenient, facile, crosslinking strategies for researchers with littleor no chemistry experience in preparing bioconjugates.

The EETI-II knottin protein (SEQ ID NO: 39 from U.S. Pat. No. 8,536,301,the contents of which are incorporated herein by reference) contains adisulfide knotted topology and possesses multiple solvent-exposed loopsthat are amenable to mutagenesis. Some embodiments use EETI-II as themolecular scaffold.

Another example of a knottin protein which can be used as a molecularscaffold is AgRP or agatoxin. The amino acid sequences of AgRP (SEQ IDNO: 40 from U.S. Pat. No. 8,536,301) and agatoxin (SEQ ID NO: 41 fromU.S. Pat. No. 8,536,301) differ but their structure is identical.Exemplary AgRP knottins are found in Table 1 from U.S. Pat. No.8,536,301.

Additional AgRP engineered knottins can be made as described in theabove-referenced US 2009/0257952 to Cochran et al. (the contents ofwhich are incorporated herein by reference). AgRP knottin fusions can beprepared using AgRP loops 1, 2 and 3, as well as loop 4.

The present polypeptides may be produced by recombinant DNA or may besynthesized in solid phase using a peptide synthesizer, which has beendone for the peptides of all three scaffolds described herein. They mayfurther be capped at their N-termini by reaction with fluoresceinisothiocyanate (FITC) or other labels, and, still further, may besynthesized with amino acid residues selected for additionalcrosslinking reactions. TentaGel S RAM Fmoc resin (Advanced ChemTech)may be used to give a C-terminal amide upon cleavage. B-alanine is usedas the N-terminal amino acid to prevent thiazolidone formation andrelease of fluorescein during peptide deprotection (Hermanson, 1996).Peptides are cleaved from the resin and side-chains are deprotected with8% trifluoroacetic acid, 2% triisopropylsilane, 5% dithiothreitol, andthe final product is recovered by ether precipitation. Peptides arepurified by reverse phase HPLC using an acetonitrile gradient in 0.1%trifluoroacetic acid and a C4 or C18 column (Vydac) and verified usingmatrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF) or electrospray ionization-mass spectrometry(ESI-MS).

When the present peptides are produced by recombinant DNA, expressionvectors encoding the selected peptide are transformed into a suitablehost. The host should be selected to ensure proper peptide folding anddisulfide bond formation as described above. Certain peptides, such asEETI-II, can fold properly when expressed in prokaryotic hosts such asbacteria.

Dimeric, trimeric, and tetrameric complexes of the present peptides canbe formed through genetic engineering of the above sequences or byreaction of the synthetic cross-linkers with engineered peptidescarrying an introduced cysteine residue, for example on the C-terminusof the peptide. These oligomeric peptide complexes can be purified bygel filtration. Oligomers of the present peptides can be prepared bypreparing vectors encoding multiple peptide sequences end-to-end. Also,multimers may be prepared by complexing the peptides, such as, e.g.,described in U.S. Pat. No. 6,265,539. There, an active HJV peptide isprepared in multimer form by altering the amino-terminal residue of thepeptide so that it is peptide-bonded to a spacer peptide that containsan amino-terminal lysyl residue and one to about five amino acidresidues such as glycyl residues to form a composite polypeptide.Alternatively, each peptide is synthesized to contain a cysteine (Cys)residue at each of its amino- and carboxy-termini. The resultingdi-cysteine-terminated (di-Cys) peptide is then oxidized to polymerizethe di-Cys peptide monomers into a polymer or cyclic peptide multimer.Multimers may also be prepared by solid phase peptide synthesisutilizing a lysine core matrix. The present peptides may also beprepared as nanoparticles. See, “Multivalent Effects of RGD PeptidesObtained by Nanoparticle Display,” Montet, et al., J. Med. Chem.; 2006;49(20) pp 6087-6093. EETI dimerization may be carried out with thepresent EETI-II peptides according to the EETI-II dimerization paper:“Grafting of thrombopoietin-mimetic peptides into cystine knotminiproteins yields high-affinity fhrombopoietin antagonist andagonists,” Krause, et al., FEBS Journal; 2006; 274 pp 86-95. This isfurther described in PCT application No. PCT/US2013/065610, hereinincorporated by reference.

Synergistic sites on fibronectin and other adhesion proteins have beenidentified for enhanced integrin binding (Ruoslahti, 1996; Koivunen etal., 1994; Aota et al., 1994; Healy et al., 1995). The ability toincorporate different integrin-specific motifs into one soluble moleculewould have an important impact on therapeutic development. Crosslinkerswith heterofunctional specificity may be used for creatingintegrin-binding proteins with synergistic binding effects. In addition,these same crosslinkers could easily be used to create bispecifictargeting molecules, or as vehicles for delivery of radionuclides ortoxic agents for therapeutic applications.

3. Integrin-Binding Polypeptides

The integrin-binding polypeptides for use in Fc fusions include anintegrin-binding loop (e.g., RGD peptide sequence) and a knottinpolypeptide scaffold. Such integrin-binding polypeptides are describedin U.S. Pat. No. 8,536,301, the contents of which are incorporatedherein by reference. As described in U.S. Pat. No. 8,536,301,integrin-binding polypeptides may be varied in the non-RGD residues to acertain degree without affecting binding specificity and potency. Forexample, if three of the eleven residues were varied, one would haveabout 70% identity to 2.5D. Table 1 shows exemplary integrin-bindingpolypeptides within the scope of the invention, and their specificknottin polypeptide scaffold (e.g., EETI-II or AgRP). In someembodiments, integrin-binding polypeptides for use in Fc fusions arepeptides 2.5F and 2.5FmodK, as described herein(GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG, 2.5F, SEQ ID NO:130 andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG, 2.5FmodK, SEQ ID NO:131), as well asGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), and/orGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135).

In certain embodiments, the integrin-binding polypeptide binds to_(v)β₃, α_(v)β₅, or α5β1 separately.

In certain embodiments, the integrin-binding polypeptide binds to _(v)β₃and _(v)β₅ simultaneously.

In certain embodiments, the integrin-binding polypeptide binds to_(v)β₃, α_(v)β₅, and α5β₁ simultaneously.

In certain embodiments, the integrin-binding polypeptide is 2.5F or2.5FmodK, as described herein (GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG, 2.5F,SEQ ID NO:130 and GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG, 2.5FmodK, SEQ IDNO:131), as well as GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ IDNO:132), GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), and/orGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135). Insome embodiments, an integrin-binding polypeptide as recited in Table 1of U.S. Pat. No. 8,536,301 can also be used in Fc fusion as describedherein.

The present polypeptides target α_(v)β1, α_(v)β3, α_(v)β5, α_(v)β6, andα5β1 integrin receptors. They do not bind to other integrins tested,such as α_(iib)β3, where there was little to no affinity (as describedin US. Thus, these engineered integrin-binding polypeptides have broaddiagnostic and therapeutic applications in a variety of human cancersthat specifically overexpress the above named integrins. As describedbelow, these polypeptides bind with high affinity to bothdetergent-solubilized and tumor cell surface integrin receptors.

The α_(v)β3 (and α_(v)β5) integrins are also highly expressed on manytumor cells including osteosarcomas, neuroblastomas, carcinomas of thelung, breast, prostate, and bladder, glioblastomas, and invasivemelanomas The α_(v)β3 integrin has been shown to be expressed on tumorcells and/or the vasculature of breast, ovarian, prostate, and coloncarcinomas, but not on normal adult tissues or blood vessels. Also, theα5β1 integrin has been shown to be expressed on tumor cells and/or thevasculature of breast, ovarian, prostate, and colon carcinomas, but noton normal adult tissue or blood vessels. The present, small,conformationally-constrained polypeptides (about 33 amino acids) are soconstrained by intramolecular bonds. For example, EETI-II has threedisulfide linkages. This will make it more stable in vivo.

Until now, it is believed that the development of a single agent thatcan bind α_(v)β3, α_(v)β5, and α5β1 integrins with high affinity andspecificity has not been achieved. Since all three of these integrinsare expressed on tumors and are involved in mediating angiogenesis andmetastasis, a broad spectrum targeting agent (i.e., α_(v)β₃, α_(v)β₅,and α₅β₁) will likely be more effective for diagnostic and therapeuticapplications.

The present engineered knottin-Fc fusions have several advantages overpreviously identified integrin-targeting compounds. They possess acompact, disulfide-bonded core that confers proteolytic resistance andexceptional in vivo stability.

Our studies indicate the half-life of integrin-binding-Fc fusion proteinin mouse serum to be greater than 90 hours. Their larger size (^(˜)3-4kDa) and enhanced affinity compared to RGD-based cyclic peptides conferenhanced pharmacokinetics and biodistribution for molecular imaging andtherapeutic applications. These integrin-binding-Fc fusion proteins aresmall enough to allow for chemical synthesis and site-specificconjugation of imaging probes, radioisotopes, or chemotherapeuticagents. Furthermore, they can easily be chemically modified to furtherimprove in vivo properties if necessary.

4. Integrin-Binding Polypeptide-Fc Fusion

The integrin-binding polypeptide-Fc fusions (knottin-Fc fusions)described herein and in U.S. Patent Application No. 2014/0073518, hereinincorporated by reference in its entirety, combine an engineeredintegrin-binding polypeptide (within a knottin scaffold) and an Fcdomain or antibody like construct capable of binding FcγR and inducingADCC.

The Fc portion of an antibody is formed by the two carboxy terminaldomains of the two heavy chains that make up an immunoglobin molecule.The IgG molecule contains 2 heavy chains (˜50 kDa each) and 2 lightchains (˜25 kDa each). The general structure of all antibodies is verysimilar, a small region at the tip of the protein is extremely variable,allowing millions of antibodies with slightly different tip structuresto exist. This region is known as the hypervariable region (Fab). Theother fragment contains no antigen-binding activity but was originallyobserved to crystallize readily, and for this reason was named the Fcfragment, for Fragment crystallizable. This fragment corresponds to thepaired C3/4 and C3/4 domains and is the part of the antibody moleculethat interacts with effector molecules and cells. The functionaldifferences between heavy-chain isotypes lie mainly in the Fc fragment.The hinge region that links the Fc and Fab portions of the antibodymolecule is in reality a flexible tether, allowing independent movementof the two Fab arms, rather than a rigid hinge. This has beendemonstrated by electron microscopy of antibodies bound to haptens. Thusthe present fusion proteins can be made to contain two knottin peptides,one on each arm of the antibody fragment.

The Fc portion varies between antibody classes (and subclasses) but isidentical within that class. The C-terminal end of the heavy chain formsthe Fc region. The Fc region plays an important role as a receptorbinding portion. The Fc portion of antibodies will bind to Fc receptorsin two different ways. For example, after IgG and IgM bind to a pathogenby their Fab portion their Fc portions can bind to receptors onphagocytic cells (like macrophages) inducing phagocytosis.

The present integrin-binding polypeptide-Fc fusions can be implementedsuch that the Fc portion is used to provide dual binding capability,and/or for half-life extension, for improving expression levels, etc.The Fc fragment in the integrin-binding polypeptide-Fc fusion can be,for example, from murine IgG2a or human IgG1. In some embodiments, theFc fragment can be from mouse IgG1, IgG2, IgG3, or mouse IgG4, as wellas variants thereof. In some embodiments, the Fc fragment can be fromhuman IgG1, IgG2, IgG3, or mouse IgG4, as well as variants thereof. See,for example, FIG. 1. Linkers can be optionally used to connect theintegrin binding portion (knottin) to the Fc portion.

In some embodiments, the linkers do not affect the binding affinity ofthe integrin-binding polypeptide-Fc fusions to integrins or Fcreceptors. A variety of Fc domain gene sequences (e.g., mouse and humanconstant region gene sequences) are available in the form of publiclyaccessible deposits.

5. Fe-Domains

A variety of Fc domain gene sequences (e.g., mouse and human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains comprising an Fc domain sequence canbe selected lacking a particular effector function and/or with aparticular modification to reduce immunogenicity. Many sequences ofantibodies and antibody-encoding genes have been published and suitableFc domain sequences (e.g., hinge, CH₂, and/or CH₃ sequences, or portionsthereof) can be derived from these sequences using art recognizedtechniques. The genetic material obtained using any of the foregoingmethods may then be altered or synthesized to obtain polypeptides usedherein. It will further be appreciated that alleles, variants andmutations of constant region DNA sequences are suitable for use in themethods disclosed herein.

Integrin-binding polypeptide-Fc fusions suitable for use in the methodsdisclosed herein may comprise one or more Fc domains (e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, or more Fc domains). In some embodiments, the Fc domainsmay be of different types. In some embodiments, at least one Fc domainpresent in an integrin-binding polypeptide-Fc fusion comprises a hingedomain or portion thereof. In another embodiment, an integrin-bindingpolypeptide-Fc fusion comprises at least one Fc domain which comprisesat least one CH2 domain or portion thereof. In another embodiment, anintegrin-binding polypeptide-Fc fusion comprises at least one Fc domainwhich comprises at least one CH₃ domain or portion thereof. In anotherembodiment, an integrin-binding polypeptide-Fc fusion comprises at leastone Fc domain which comprises at least one CH₄ domain or portionthereof. In another embodiment, an integrin-binding polypeptide-Fcfusion comprises at least one Fc domain which comprises at least onehinge domain or portion thereof and at least one CH₂ domain or portionthereof (e.g., in the hinge-CH₂ orientation). In another embodiment, anintegrin-binding polypeptide-Fc fusion comprises at least one Fc domainwhich comprises at least one CH₂ domain or portion thereof and at leastone CH₃ domain or portion thereof (e.g., in the CH₂—CH₃ orientation). Inanother embodiment, an integrin-binding polypeptide-Fc fusion comprisesat least one Fc domain comprising at least one hinge domain or portionthereof, at least one CH₂ domain or portion thereof, and least one CH₃domain or portion thereof, for example in the orientation hinge-CH₂—CH₃,hinge-CH₃—CH₂, or CH₂—CH₃-hinge.

In some embodiments, an integrin-binding polypeptide-Fc fusion comprisesat least one complete Fc region derived from one or more immunoglobulinheavy chains (e.g., an Fc domain including hinge, CH₂, and CH₃ domains,although these need not be derived from the same antibody). In otherembodiments an integrin-binding polypeptide-Fc fusion comprises at leasttwo complete Fc domains derived from one or more immunoglobulin heavychains. In certain embodiments, the complete Fc domain is derived from ahuman IgG immunoglobulin heavy chain (e.g., human IgG1).

In another embodiment, an integrin-binding polypeptide-Fc fusioncomprises at least one Fc domain comprising a complete CH₃ domain. Inanother embodiment, an integrin-binding polypeptide-Fc fusion comprisesat least one Fc domain comprising a complete CH₂ domain. In anotherembodiment, an integrin-binding polypeptide-Fc fusion comprises at leastone Fc domain comprising at least a CH₃ domain, and at least one of ahinge region, and a CH₂ domain. In one embodiment, an integrin-bindingpolypeptide-Fc fusion comprises at least one Fc domain comprising ahinge and a CH₃ domain. In another embodiment, an integrin-bindingpolypeptide-Fc fusion comprises at least one Fc domain comprising ahinge, a CH₂, and a CH₃ domain. In some embodiments, the Fc domain isderived from a human IgG immunoglobulin heavy chain (e.g., human IgG1).In some embodiments, a human IgG1 Fc domain is used with a hinge regionmutation, substitution, or deletion to remove or substitute one or morehinge region cysteine residues.

The constant region domains or portions thereof making up an Fc domainof an integrin-binding polypeptide-Fc fusion may be derived fromdifferent immunoglobulin molecules. For example, a polypeptide used inthe invention may comprise a CH₂ domain or portion thereof derived froman IgG1 molecule and a CH₃ region or portion thereof derived from anIgG3 molecule. In some embodiments, an integrin-binding polypeptide-Fcfusion can comprise an Fc domain comprising a hinge domain derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. As setforth herein, it will be understood by one of ordinary skill in the artthat an Fc domain may be altered such that it varies in amino acidsequence from a naturally occurring antibody molecule.

In other constructs it may be desirable to provide a peptide spacerbetween one or more constituent Fc domains. For example, in someembodiments, a peptide spacer may be placed between a hinge region and aCH₂ domain and/or between a CH₂ and a CH₃ domain. For example,compatible constructs could be expressed wherein the CH₂ domain has beendeleted and the remaining CH₃ domain (synthetic or unsynthetic) isjoined to the hinge region with a 1-20, 1-10, or 1-5 amino acid peptidespacer. Such a peptide spacer may be added, for instance, to ensure thatthe regulatory elements of the constant region domain remain free andaccessible or that the hinge region remains flexible. Preferably, anylinker peptide compatible with the instant invention will be relativelynon-immunogenic and not prevent proper folding of the Fc.

6. Changes to Fc Amino Acids

In some embodiments, an Fc domain is altered or modified, e.g., by aminoacid mutation (e.g., addition, deletion, or substitution). As usedherein, the term “Fc domain variant” refers to an Fc domain having atleast one amino acid modification, such as an amino acid substitution,as compared to the wild-type Fc from which the Fc domain is derived. Forexample, wherein the Fc domain is derived from a human IgG1 antibody, avariant comprises at least one amino acid mutation (e.g., substitution)as compared to a wild type amino acid at the corresponding position ofthe human IgG1 Fc region.

In some embodiments, the hinge region of human IgG1 Fc domain is alteredby an amino acid substitution or deletion to mutate or remove one ormore of three hinge region cysteine residues (located at residues 220,226, and 229 by EU numbering). In some aspects, the upper hinge regionis deleted to remove a cysteine that pairs with the light chain. Forexample, in some embodiments, amino acids “EPKSC” in the upper hingeregion are deleted, as set forth in SEQ ID NO: 3 from U.S. Pat. No.8,536,301. In other aspects, one or more of three hinge region cysteinesis mutated (e.g., to serine). In certain embodiments, cysteine 220 ismutated to serine.

In some embodiments, the Fc variant comprises a substitution at an aminoacid position located in a hinge domain or portion thereof. In someembodiments, the Fc variant comprises a substitution at an amino acidposition located in a CH₂ domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH₃ domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH₄ domain or portion thereof.

In some embodiments, an integrin-binding polypeptide-Fc fusion comprisesan Fc variant comprising more than one amino acid substitution. The anintegrin-binding polypeptide-Fc fusion used in the methods describedherein may comprise, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acid substitutions.

In some embodiments, the amino acid substitutions are spatiallypositioned from each other by an interval of at least 1 amino acidposition or more, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid positions or more. In some embodiments, the engineered aminoacids are spatially positioned apart from each other by an interval ofat least 5, 10, 15, 20, or 25 amino acid positions or more.

In some embodiments, an integrin-binding polypeptide-Fc fusion comprisesan amino acid substitution to an Fc domain which alters theantigen-independent effector functions of the polypeptide, in particularthe circulating half-life of the polypeptide.

In one embodiment, the integrin-binding polypeptide-Fc fusion exhibitsenhanced binding to an activating FcyR (e.g. FcγI, Fcγ1α, or FcyRIIIα).Exemplary amino acid substitutions which altered FcR or complementbinding activity are disclosed in International PCT Publication No. WO2005/063815 which is incorporated by reference herein. In certainembodiments the Fc region contains at least one of the followingmutations: S239D, S239E, L261A, H268D, S298A, A330H, A330L, I332D,I332E, I332Q, K334V, A378F, A378K, A378W, A378Y, H435S, or H435G. Incertain embodiments, the Fc region contains at least one of thefollowing mutations: S239D, S239E, I332D or I332E or H268D. In certainembodiments, the Fc region contains at least one of the followingmutations: I332D or I332E or H268D.

The integrin-binding polypeptide-Fc fusion used herein may also comprisean amino acid substitution which alters the glycosylation of theintegrin-binding polypeptide-Fc fusion. For example, the Fc domain ofthe integrin-binding polypeptide-Fc fusion may comprise an Fc domainhaving a mutation leading to reduced glycosylation (e.g., N- or O-linkedglycosylation) or may comprise an altered glycoform of the wild-type Fcdomain (e.g., a low fucose or fucose-free glycan). In anotherembodiment, the integrin-binding polypeptide-Fc fusion has an amino acidsubstitution near or within a glycosylation motif, for example, anN-linked glycosylation motif that contains the amino acid sequence NXTor NXS. Exemplary amino acid substitutions which reduce or alterglycosylation are disclosed in WO 05/018572 and US 2007/0111281, whichare incorporated by reference herein. In other embodiments, theintegrin-binding polypeptide-Fc fusion used herein comprises at leastone Fc domain having engineered cysteine residue or analog thereof whichis located at the solvent-exposed surface. In some embodiments, theintegrin-binding polypeptide-Fc fusion used herein comprises an Fcdomain comprising at least one engineered free cysteine residue oranalog thereof that is substantially free of disulfide bonding with asecond cysteine residue. Any of the above engineered cysteine residuesor analogs thereof may subsequently be conjugated to a functional domainusing art-recognized techniques (e.g., conjugated with a thiol-reactiveheterobifunctional linker).

In one embodiment, the integrin-binding polypeptide-Fc fusion usedherein may comprise a genetically fused Fc domain having two or more ofits constituent Fc domains independently selected from the Fc domainsdescribed herein. In one embodiment, the Fc domains are the same. Inanother embodiment, at least two of the Fc domains are different. Forexample, the Fc domains of the integrin-binding polypeptide-Fc fusionused herein comprise the same number of amino acid residues or they maydiffer in length by one or more amino acid residues (e.g., by about 5amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about10 residues, about 15 residues, about 20 residues, about 30 residues,about 40 residues, or about 50 residues). In some embodiments, the Fcdomains of the integrin-binding polypeptide-Fc fusion used herein maydiffer in sequence at one or more amino acid positions. For example, atleast two of the Fc domains may differ at about 5 amino acid positions(e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about15 positions, about 20 positions, about 30 positions, about 40positions, or about 50 positions).

VIII. Nucleic Acid Compositions

Nucleic acid compositions encoding the integrin-binding polypeptide-Fcfusions of the invention are also provided, as well as expressionvectors containing the nucleic acids and host cells transformed with thenucleic acid and/or expression vector compositions.

The nucleic acid compositions that encode the integrin-bindingpolypeptide-Fc are generally put into a single expression vectors isknown in the art, transformed into host cells, where they are expressedto form the integrin-binding polypeptide-Fc of the invention. Thenucleic acids can be put into expression vectors that contain theappropriate transcriptional and translational control sequences,including, but not limited to, signal and secretion sequences,regulatory sequences, promoters, origins of replication, selectiongenes, etc.

For example, to express the protein DNA, DNAs can be obtained bystandard molecular biology techniques (e.g., PCR amplification or genesynthesis) and the DNAs can be inserted into expression vectors suchthat the genes are operatively linked to transcriptional andtranslational control sequences. In this context, the term “operativelylinked” is intended to mean that an antibody gene is ligated into avector such that transcriptional and translational control sequenceswithin the vector serve their intended function of regulating thetranscription and translation of the antibody gene. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. The protein genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the gene fragment and vector, or blunt end ligationif no restriction sites are present). Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the protein (including fusion proteins) from ahost cell. The gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the gene. The signalpeptide can be an immunoglobulin signal peptide or a heterologous signalpeptide (i.e., a signal peptide from a non-immunoglobulin protein).Exemplary signal peptides include but are not limited toMTRLTVLALLAGLLASSRA (SEQ ID NO:160).

In addition to the protein genes, the recombinant expression vectorsaccording to at least some embodiments of the invention carry regulatorysequences that control the expression of the genes in a host cell. Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals)that control the transcription or translation of the genes. Suchregulatory sequences are described, for example, in Goeddel (“GeneExpression Technology”, Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SR α. promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the protein genes and regulatory sequences, therecombinant expression vectors according to at least some embodiments ofthe invention may carry additional sequences, such as sequences thatregulate replication of the vector in host cells (e.g., origins ofreplication) and selectable marker genes. The selectable marker genefacilitates selection of host cells into which the vector has beenintroduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the proteins of the invention, an expression vectorencoding the protein is transfected into a host cell by standardtechniques. The various forms of the term “transfection” are intended toencompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the proteins according to at least some embodimentsof the invention in either prokaryotic or eukaryotic host cells,expression of antibodies in eukaryotic cells, and most preferablymammalian host cells, is the most preferred.

In some embodiments, mammalian host cells for expressing the recombinantproteins include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSOmyeloma cells, COS cells and SP2 cells. In particular, for use with NSOmyeloma cells, another preferred expression system is the GS geneexpression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.When recombinant expression vectors encoding protein genes areintroduced into mammalian host cells, the proteins are produced byculturing the host cells for a period of time sufficient to allow forexpression of the protein in the host cells or, more preferably,secretion of the protein into the culture medium in which the host cellsare grown.

IX. Immune Checkpoint Modulators

In certain embodiments, immune checkpoint modulators (inhibitors orstimulators) are used in combination with other therapeutic agentsdescribed herein (e.g., IL-2, extended-PK IL-2, INFα, and/or integrinbinding-Fc fusion proteins). T cell activation and effector functionsare balanced by co-stimulatory and inhibitory signals, referred to as“immune checkpoints.” Inhibitory ligands and receptors that regulate Tcell effector functions are overexpressed on tumor cells. Subsequently,agonists of co-stimulatory receptors or antagonists of inhibitorysignals, result in the amplification of antigen-specific T cellresponses.

1. Immune Checkpoint Inhibitors

In certain embodiments, the immune checkpoint modulator is an immunecheckpoint inhibitor. In contrast to therapeutic antibodies which targettumor cells directly, immune checkpoint inhibitors enhance endogenousanti-tumor activity. In certain embodiments, the immune checkpointinhibitor suitable for use in the methods disclosed herein, is anantagonist of inhibitory signals, e.g., an antibody which targets, forexample, PD-1, PD-L1, CTLA-4, and B7-H3, B7-H4. These ligands andreceptors are reviewed in Pardoll, D., Nature. 12: 252-264, 2012.

In some embodiments, the immune checkpoint inhibitor is selected fromthe group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody,and an anti-CTLA-4 antibody. In some embodiments, the immune checkpointinhibitor is an anti-PD-1 antibody. In some embodiments, the immunecheckpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, theimmune checkpoint inhibitor is an anti-CTLA-4 antibody. In someembodiments, NOD201 is used in combination with anti-PD-1 antibody andIL-2. In some embodiments, NOD201 is used in combination with anti-PD-1antibody and low dose IL-2. In some embodiments, NOD201 is used incombination with a checkpoint stimulator and INFα. In some embodiments,NOD201 is not used in combination with an anti-TIGIT antibody, ananti-LAG-3 antibody, or an anti-TIM-3 antibody. In some embodiments,NOD201 is not used in combination with an anti-TIGIT antibody. In someembodiments, NOD201 is not used in combination with an anti-LAG-3antibody. In some embodiments, NOD201 is not used in combination with ananti-TIM-3 antibody. In some embodiments, the immune checkpointinhibitor is not an anti-TIGIT antibody, an anti-LAG-3 antibody, or ananti-TIM-3 antibody. In some embodiments, the immune checkpointinhibitor is not an anti-TIGIT antibody. In some embodiments, the immunecheckpoint inhibitor is not an anti-LAG-3 antibody. In some embodiments,the immune checkpoint inhibitor is not an anti-TIM-3 antibody.

Disclosed herein are methods for treating a subject afflicted withdiseases such as cancer, which methods comprise administering to thesubject a composition comprising a therapeutically effective amount ofan integrin-binding-Fc fusion protein as described herein. In someembodiments, the method comprising administering an integrin-binding-Fcfusion protein such as NOD201, NOD203, and/or NOD204, as well ascombinations thereof. In some embodiments, the integrin-binding-Fcfusion protein is combined with molecule which blocks the immunecheckpoint, and an integrin-binding-Fc fusion protein. In someembodiments, the methods for treating a subject afflicted with diseasessuch as cancer, which methods comprise administering to the subject acomposition comprising a therapeutically effective amount of a moleculewhich blocks the immune checkpoint, an integrin-binding-Fc fusionprotein, and IL-2 (e.g., wild-type IL-2, Proleukin, and/or extended-PKIL-2). In some embodiments, the immune checkpoint inhibitor is anantibody or an antigen-binding portion thereof, that disrupts orinhibits signaling from an inhibitory immunoregulator. In someembodiments, the immune checkpoint inhibitor is a small molecule thatdisrupts or inhibits signaling from an inhibitory immunoregulator.

In some embodiments, the inhibitory immunoregulator (immune checkpointinhibitor) is a component of the PD-1/PD-L1 signaling pathway.Accordingly, some embodiments provide methods for immunotherapy of asubject afflicted with cancer, which methods comprise administering tothe subject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that disrupts the interaction betweenthe PD-1 receptor and its ligand, PD-L1. Antibodies known in the artwhich bind to PD-1 and disrupt the interaction between the PD-1 and itsligand, PD-L1, and stimulates an anti-tumor immune response, aresuitable for use in the methods disclosed herein. In some embodiments,the antibody or antigen-binding portion thereof binds specifically toPD-1. For example, antibodies that target PD-1 and which can find usedin the methods of the present invention include, e.g., but are notlimited to nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab(lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibodyJS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro,Inc.), Pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1monoclonal Antibody BGB-A317 (BeiGene), and/or anti-PD-1 antibodySHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810(Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb),and/or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In someembodiments, the PD-1 antibody is from clone: RMP1-14 (rat IgG)—BioXcellcat #BP0146. Other suitable antibodies for use in the methods disclosedherein are anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449,herein incorporated by reference. In some embodiments, the antibody orantigen-binding portion thereof binds specifically to PD-L1 and inhibitsits interaction with PD-1, thereby increasing immune activity. Anyantibodies known in the art which bind to PD-L1 and disrupt theinteraction between the PD-1 and PD-L1, and stimulates an anti-tumorimmune response, are suitable for use in the methods disclosed herein.For example, antibodies that target PD-L1 and are in clinical trials,include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech).Other suitable antibodies that target PD-L1 are disclosed in U.S. Pat.No. 7,943,743, herein incorporated by reference. It will be understoodby one of ordinary skill that any antibody which binds to PD-1 or PD-L1,disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immuneresponse, are suitable for use in the methods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe CTLA-4 signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets CTLA-4 and disrupts its interaction with CD80 and CD86.Exemplary antibodies that target CTLA-4 include ipilimumab (MDX-010,MDX-101, Bristol-Myers Squibb), which is FDA approved, and tremelimumab(ticilimumab, CP-675, 206, Pfizer), currently undergoing human trials.Other suitable antibodies that target CTLA-4 are disclosed in WO2012/120125, U.S. Pat. Nos. 6,984,720, 6,682,7368, and U.S. PatentApplications 2002/0039581, 2002/0086014, and 2005/0201994, hereinincorporated by reference. It will be understood by one of ordinaryskill that any antibody which binds to CTLA-4, disrupts its interactionwith CD80 and CD86, and stimulates an anti-tumor immune response, aresuitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe LAG-3 (lymphocyte activation gene 3) signaling pathway. Accordingly,certain embodiments provide methods for immunotherapy of a subjectafflicted with cancer, which methods comprise administering to thesubject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that targets LAG-3 and disrupts itsinteraction with MHC class II molecules. An exemplary antibody thattargets LAG-3 is IMP321 (Immutep), currently undergoing human trials.Other suitable antibodies that target LAG-3 are disclosed in U.S. PatentApplication 2011/0150892, herein incorporated by reference. It will beunderstood by one of ordinary skill that any antibody which binds toLAG-3, disrupts its interaction with MHC class II molecules, andstimulates an anti-tumor immune response, are suitable for use in themethods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe B7 family signaling pathway. In some embodiments, the B7 familymembers are B7-H3 and B7-H4. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets B7-H3 or -H4. The B7 family does not have any definedreceptors but these ligands are upregulated on tumor cells ortumor-infiltrating cells. Preclinical mouse models have shown thatblockade of these ligands can enhance anti-tumor immunity. An exemplaryantibody that targets B7-H3 is MGA271 (Macrogenics), currentlyundergoing human trials. Other suitable antibodies that target B7 familymembers are disclosed in U.S. Patent Application 2013/0149236, hereinincorporated by reference. It will be understood by one of ordinaryskill that any antibody which binds to B7-H3 or H4, and stimulates ananti-tumor immune response, are suitable for use in the methodsdisclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe TIM-3 (T cell membrane protein 3) signaling pathway. Accordingly,certain embodiments provide methods for immunotherapy of a subjectafflicted with cancer, which methods comprise administering to thesubject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that targets TIM-3 and disrupts itsinteraction with galectin 9. Suitable antibodies that target TIM-3 aredisclosed in U.S. Patent Application 2013/0022623, herein incorporatedby reference. It will be understood by one of ordinary skill that anyantibody which binds to TIM-3, disrupts its interaction with galectin 9,and stimulates an anti-tumor immune response, are suitable for use inthe methods disclosed herein.

It should be understood that antibodies targeting immune checkpointssuitable for use in the methods disclosed herein are not limited tothose described herein. Moreover, it will be understood by one ofordinary skill in the art that other immune checkpoint targets can alsobe targeted by antagonists or antibodies in the methods describedherein, provided that the targeting results in the stimulation of ananti-tumor immune response as reflected in, e.g., an increase in T cellproliferation, enhanced T cell activation, and/or increased cytokineproduction (e.g., IFN-γ, IL-2).

2. Immune Checkpoint Stimulators

In certain embodiments, the immune checkpoint modulator is an immunecheckpoint stimulator. In contrast to therapeutic antibodies whichtarget tumor cells directly, immune checkpoint stimulators enhanceendogenous immune system activity and/or reduce endogenous immune systemsuppression activity. In certain embodiments, the immune checkpointstimulator suitable for use in the methods disclosed herein, is anagonist of stimulatory signals or an antagonist of suppression signals,e.g., an antibody which targets, for example, 4-1BB/CD137, IFNα, GITR,and OX40. These ligands and receptors are reviewed in Peggs, K. S., etal., Clin Exp Immunol., 157(1): 9-19 (2009).

In some embodiments, the immune checkpoint stimulator is selected fromthe group consisting of an anti-4-1BB/CD137 antibody, an anti-IFNαantibody, an anti-GITR antibody, and an anti-OX40 antibody. In someembodiments, the immune checkpoint stimulator is an anti-4-1BB/CD137antibody. In some embodiments, the immune checkpoint stimulator is ananti-IFNα antibody. In some embodiments, the immune checkpointstimulator is an anti-GITR antibody. In some embodiments, the immunecheckpoint stimulator is an anti-OX40 antibody. In some embodiments,NOD201 is used in combination with a checkpoint stimulator and IL-2. Insome embodiments, NOD201 is used in combination with a checkpointstimulator and low dose IL-2. In some embodiments, NOD201 is used incombination with a checkpoint stimulator and INFα. In some embodiments,NOD201 is used in combination with IFNα.

Disclosed herein are methods for treating a subject afflicted withdiseases such as cancer, which methods comprise administering to thesubject a composition comprising a therapeutically effective amount ofan integrin-binding-Fc fusion protein as described herein. In someembodiments, the method comprising administering an integrin-binding-Fcfusion protein such as NOD201, NOD203, and/or NOD204, as well ascombinations thereof. In some embodiments, the integrin-binding-Fcfusion protein is combined with molecule which blocks the immunecheckpoint, and an integrin-binding-Fc fusion protein. In someembodiments, the methods for treating a subject afflicted with diseasessuch as cancer, which methods comprise administering to the subject acomposition comprising a therapeutically effective amount of a moleculewhich enhances the immune checkpoint, enhances the immune system, and/orreduces immune system suppression, an integrin-binding-Fc fusionprotein, and IL-2 (e.g., wild-type IL-2, Proleukin, and/or extended-PKIL-2). In some embodiments, the immune checkpoint stimulator is anantibody or an antigen-binding portion thereof, that enhances orincreases signaling from a stimulatory immunoregulator. In someembodiments, the immune checkpoint stimulator is an antibody or anantigen-binding portion thereof, that disrupts or inhibits signalingfrom a suppressive immunoregulator. In some embodiments, the immunecheckpoint inhibitor is a small molecule that enhances or increasessignaling from a stimulatory immunoregulator. In some embodiments, theimmune checkpoint inhibitor is a small molecule that disrupts orinhibits signaling from a suppressive immunoregulator.

In some embodiments, the inhibitory immunoregulator is a component ofthe 4-1BB/CD137 signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets 4-1BB/CD137 and disrupts its interaction with CD137L. Itwill be understood by one of ordinary skill that any antibody whichbinds to 4-1BB/CD137, disrupts its interaction with CD137L or anotherligand, and stimulates an anti-tumor immune response or an immunestimulatory response that results in anti-tumor activity overall, aresuitable for use in the methods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe IFNα signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets IFNα and disrupts its interaction with its ligand. It willbe understood by one of ordinary skill that any antibody which binds toIFNα, disrupts its interaction with its ligand, and stimulates ananti-tumor immune response or an immune stimulatory response thatresults in anti-tumor activity overall, are suitable for use in themethods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe GITR signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets GITR and disrupts its interaction with its ligand. It willbe understood by one of ordinary skill that any antibody which binds toGITR, disrupts its interaction with GITRL or another ligand, andstimulates an anti-tumor immune response or an immune stimulatoryresponse that results in anti-tumor activity overall, are suitable foruse in the methods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe OX40 (CD134) signaling pathway. Accordingly, some embodimentsprovide methods for immunotherapy of a subject afflicted with cancer,which methods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets OX40 and disrupts its interaction with its ligand. It willbe understood by one of ordinary skill that any antibody which binds toOX40, disrupts its interaction with OX40L or another ligand, andstimulates an anti-tumor immune response or an immune stimulatoryresponse that results in anti-tumor activity overall, are suitable foruse in the methods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe CD40 signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets CD40 and disrupts its interaction with its ligand. It willbe understood by one of ordinary skill that any antibody which binds toCD40, disrupts its interaction with its ligand, and stimulates ananti-tumor immune response or an immune stimulatory response thatresults in anti-tumor activity overall, are suitable for use in themethods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe ICOS signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets ICOS and disrupts its interaction with its ligand. It willbe understood by one of ordinary skill that any antibody which binds toICOS, disrupts its interaction with its ligand, and stimulates ananti-tumor immune response or an immune stimulatory response thatresults in anti-tumor activity overall, are suitable for use in themethods disclosed herein.

In some embodiments, the inhibitory immunoregulator is a component ofthe CD28 signaling pathway. Accordingly, some embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets CD28 and disrupts its interaction with its ligand. It willbe understood by one of ordinary skill that any antibody which binds toCD28, disrupts its interaction with its ligand, and stimulates ananti-tumor immune response or an immune stimulatory response thatresults in anti-tumor activity overall, are suitable for use in themethods disclosed herein.

It should be understood that antibodies targeting immune checkpointssuitable for use in the methods disclosed herein are not limited tothose described herein. Moreover, it will be understood by one ofordinary skill in the art that other immune checkpoint targets can alsobe targeted by antagonists or antibodies in the methods describedherein, provided that the targeting results in the stimulation of animmune response as reflected in, e.g., an increase in T cellproliferation, enhanced T cell activation, and/or increased cytokineproduction (e.g., IFN-γ, IL-2).

X. Alternatives to Immune Checkpoint Modulators

In certain embodiments, an antagonist of vascular endothelial growthfactor (VEGF) is used in place of an immune checkpoint inhibitor. VEGFhas recently been demonstrated to play a role in immune suppression(Liang, W.-C. et al. J. Biol. Chem. (2006) Vol 281: 951-961; Voron, T.et al. Front Oncol (2014) Vol. 4: Article 70; Terme, M. et al, Clin DevImmunol (2012) Vol. 2012: Article ID 492920; Kandalaft, E. et &\., CurrTop Microbiol Immunol (2011) Vol 344: 129-48), therefore blocking itsactivity would enhance the immune response, similar to that of an immunecheckpoint inhibitor. A “VEGF antagonist” refers to a molecule capableof neutralizing, blocking, inhibiting, abrogating, reducing orinterfering with VEGF activities including its binding to one or moreVEGF receptors. Non-limiting examples of VEGF antagonists includeanti-VEGF antibodies and antigen-binding fragments thereof, receptormolecules and derivatives which bind specifically to VEGF therebysequestering its binding to one or more receptors (e.g., a VEGFreceptor), anti-VEGF receptor antibodies, VEGF receptor antagonists suchas small molecule inhibitors of the VEGFR tyrosine kinases, or adominant negative VEGF.

In certain embodiments, the VEGF antagonist is an antibody. An“anti-VEGF antibody” is an antibody that binds to VEGF with sufficientaffinity and specificity. Non-limiting examples of anti-VEGF antibodiesare described in U.S. Pat. Nos. 6,884,879, 7,060,269, 6,582,959,6,703,030, 6,054,297, US Patent Application Nos. 2006009360,20050186208, 20030206899, 20030190317, 20030203409, 20050112126, and PCTPublication Nos. WO 98/45332, 96/30046, 94/10202, 05/044853, 13/181452.The contents of these patents and patent applications are hereinincorporated by reference. In certain embodiments the VEGF antibody isbevacizumab (Avastin® Genentech/Roche) or ranibizumab (Lucentis®Genentech/Roche).

VEGF receptors, or fragments thereof, that specifically bind to VEGF canbe used to bind to and sequester the VEGF protein, thereby preventing itfrom activating downstream signaling. In certain embodiments, the VEGFreceptor, or VEGF binding fragment thereof, is a soluble VEGF receptor,such as sFlt-1. The soluble form of the receptor exerts an inhibitoryeffect on the biological activity of VEGF by binding to VEGF, therebypreventing it from binding to its natural receptors present on thesurface of target cells. Non-limiting examples of VEGF antagonists whichbind the VEGF receptor are disclosed in PCT Application Nos. 97/44453,05/000895 and U.S. Patent Application No. 20140057851. In certainembodiments the VEGF antagonist is a polypeptide with a bifunctionalsingle-chain antagonistic human VEGF variant comprising a modified VEGFwherein the modified VEGF comprises a loop with an integrin-recognitionRGD sequence, as described in U.S. Pat. No. 8,741,839, hereinincorporated by reference.

In certain embodiments, the VEGF antagonist binds to the VEGF receptor,and can include an antibody or VEGF fragment.

XI. Linkers

In certain embodiments, the extended-PK group is optionally fused toIL-2 via a linker. In certain embodiments, an integrin-bindingpolypeptide is fused to an Fc fragment via a linker. Suitable linkersare well known in the art, such as those disclosed in, e.g.,US2010/0210511 US2010/0179094, and US2012/0094909, which are hereinincorporated by reference in its entirety. Exemplary linkers includegly-ser polypeptide linkers, glycine-proline polypeptide linkers, andproline-alanine polypeptide linkers. In a certain embodiment, the linkeris a gly-ser polypeptide linker, i.e., a peptide that consists ofglycine and serine residues.

Exemplary gly-ser polypeptide linkers comprise the amino acid sequenceSer(Gly₄Ser)_(n), as well as (Gly₄Ser)_(n) and/or (Gly₄Ser₃)_(n). Insome embodiments, n=1. In some embodiments, n=2. In some embodiments,n=3, i.e., Ser(Gly₄Ser)₃. In some embodiments, n=4, i.e., Ser(Gly₄Ser)₄.In some embodiments, n=5. In some embodiments, n=6. In some embodiments,n=7. In some embodiments, n=8. In some embodiments, n=9. In someembodiments, n=10. Another exemplary gly-ser polypeptide linkercomprises the amino acid sequence Ser(Gly₄Ser)_(n). In some embodiments,n=1. In some embodiments, n=2. In some embodiments, n=3. In anotherembodiment, n=4. In some embodiments, n=5. In some embodiments, n=6.Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser)_(n). Insome embodiments, n=1. In some embodiments, n=2. In some embodiments,n=3. In some embodiments, n=4. In some embodiments, n=5. In someembodiments, n=6. Another exemplary gly-ser polypeptide linker comprises(Gly₃Ser)_(n). In some embodiments, n=1. In some embodiments, n=2. Insome embodiments, n=3. In some embodiments, n=4. In another embodiment,n=5. In yet another embodiment, n=6. Another exemplary gly-serpolypeptide linker comprises (Gly₄Ser3)_(n). In some embodiments, n=1.In some embodiments, n=2. In some embodiments, n=3. In some embodiments,n=4. In some embodiments, n=5. In some embodiments, n=6. Anotherexemplary gly-ser polypeptide linker comprises (Gly₃Ser)_(n). In someembodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. Insome embodiments, n=4. In another embodiment, n=5. In yet anotherembodiment, n=6.

In some embodiments, the linker polypeptide is selected from the groupconsisting of GGGGS (SEQ ID NO:136) and GGGGSGGGGSGGGGS (SEQ ID NO:137).In some embodiments, the linker polypeptide is GGGGS (SEQ ID NO:136). Insome embodiments, the linker polypeptide is GGGGSGGGGSGGGGS (SEQ IDNO:137).

XII. Other Therapeutic Agents

The integrin-binding-Fc fusion protein suitable for use in the methodsdisclosed herein, can be used in conjunction with one or moretherapeutic agents. In one embodiment, the therapeutic agent is atherapeutic antibody. In another embodiment, the therapeutic agent is atherapeutic protein. In another embodiment, the therapeutic agent is asmall molecule. In another embodiment, the therapeutic agent is anantigen. In another embodiment, the therapeutic agent is a population ofcells.

XIII. Engineered Fusion Molecules

Also provided herein are engineered molecules that comprise two or moreof IL-2, and an antibody (e.g., a therapeutic antibody, an immunecheckpoint inhibitor, or an antibody that antagonizes VEGF) or antibodyfragment described herein. Such engineered molecules can effectivelyreduce the number of components to be administered to a subject (e.g., acancer patient) in the methods described herein. In some embodiments,the antibody or antibody fragment serves as the scaffold for conjugationwith other components (e.g., IL-2).

Accordingly, in certain embodiments, the engineered molecule comprisesIL-2 and an antibody or antibody fragment. In a particular embodiment,the antibody for use in the engineered protein is a bispecific antibody,wherein one component is a therapeutic antibody and the other componentis an antibody that binds to an immune checkpoint inhibitor or anantibody that antagonizes VEGF activity. Methods for generatingbispecific antibodies are known in the art.

Accordingly, in certain embodiments, the engineered molecule comprisesIL-2 and a bispecific antibody which binds to a therapeutic target andan immune checkpoint inhibitor or an antibody that antagonizes VEGF.

In certain embodiments, the IL-2 component for use in the engineeredprotein is an IL-2 lacking a pharmacokinetic moiety (i.e., anon-extended-PK IL-2). In other embodiments, the IL-2 comprises apharmacokinetic moiety (an extended-PK IL-2).

In certain embodiments, the components of the engineered molecule areconjugated to the antibody or bispecific antibody with or without alinker. Suitable linkers for conjugation are described herein andextensively described in the art.

Regions to which polypeptide-based components (e.g., IL-2) of theengineered molecule can be fused, with or without a linker, to theantibody are generally known in the art, and include, for example, theC-terminus of the antibody heavy chain, and the C-terminus of theantibody light chain.

In certain embodiments, components of the engineered molecule do notinterfere with the function of the other components. By way of example,when the engineered protein comprises a therapeutic antibody and IL-2,the IL-2 will be fused to the therapeutic antibody in a manner such thatthe antibody retains its antigen-binding function, and IL-2 retains theability to interact with its receptor. The methods described herein,e.g., in the Examples, can be used to determine whether components ofthe engineered protein retain their respective functions.

XIV. Fusion of Integrin-Binding Polypeptides and Antibodies

In some embodiments of the present invention, the integrin-bindingpolypeptides of the present invention rather than being fused to an Fccan be fused to an antibody or binding fragment thereof, including butnot limited to single chain Fvs (ScFv) as well as Fab fragments. In someembodiments, the antibody or binding fragment thereof for fusion to theintegrin-binding polypeptide is selected from the group consisting of ananti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody ananti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-4-1-BB/CD137antibody, an anti-GITR antibody, an anti-OX40 antibody, an anti-CD40antibody, an anti-CD27 antibody, an anti-ICOS antibody, and ananti-PD-L1 antibody. In some embodiments, the integrin-bindingpolypeptide 2.5F (SEQ ID NO:130; GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG) isfused to an antibody or binding fragment thereof. In some embodiments,the integrin-binding polypeptide 2.5FmodK (SEQ ID NO:131;GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG) is fused to an antibody or bindingfragment thereof. In some embodiments, the integrin-binding polypeptideis fused to the antibody or binding fragment thereof via a linker asdescribed herein. In some embodiments, the integrin-binding polypeptideis fused to the N-terminal light chain of an antibody. In someembodiments, the integrin-binding polypeptide is fused to the C-terminallight chain of an antibody. In some embodiments, the integrin-bindingpolypeptide is fused to the N-terminal heavy chain of an antibody. Insome embodiments, the integrin-binding polypeptide is fused to theC-terminal light chain of an antibody.

In particular, antibodies for fusion to the integrin-bindingpolypeptides of the present invention include, but are not limited to,anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-1 antibodiessuch as nivolumab BMS-936558/MDX-1106/ONO-4538, AMP224, CT-011, MK-3475,anti-PD-L1 antagonistic antibodies such as BMS-936559/MDX-1105,MEDI4736, RG-7446/MPDL3280A; anti-LAG-3 such as IMP-321; agonisticantibodies targeting immunostimulatory proteins, including anti-CD40mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs(anti-4-1-BB antibodies) such as BMS-663513 urelumab (anti-4-1BBantibody; see, for example, U.S. Pat. Nos. 7,288,638 and 8,962,804,incorporated by reference herein in their entireties) and PF-05082566(utomilumab; see, for example, U.S. Pat. Nos. 8,821,867; 8,337,850; and9,468,678, as well as International Patent Application Publication No.WO 2012/032433, incorporated by reference herein in their entireties);anti-OX40 mAbs (see, for example, WO 2006/029879 or WO 2010/096418,incorporated by reference herein in their entireties); anti-GITR mAbssuch as TRX518 (see, for example, U.S. Pat. No. 7,812,135, incorporatedby reference herein in its entirety); anti-CD27 mAbs, such as varlilumabCDX-1127 (see, for example, WO 2016/145085 and U.S. Patent PublicationNos. US 2011/0274685 and US 2012/0213771, incorporated by referenceherein in their entireties) anti-ICOS mAbs (for example, MEDI-570,JTX-2011, and anti-TIM-3 antibodies (see, for example, WO 2013/006490 orU.S. Patent Publication No US 2016/0257758, incorporated by referenceherein in their entireties). Other antibodies can include monoclonalantibodies to prostate cancer, ovarian cancer, breast cancer,endometrial cancer, multiple myeloma, melanoma, lymphomas, lung cancersincluding small cell lung cancer, kidney cancer, colorectal cancer,pancreatic cancer, gastric cancer, and brain cancer (see, generallywww.clinicaltrials.gov).

XV. Methods of Making Polypeptides

In some aspects, the polypeptides described herein (e.g., IL-2, such asextended-PK IL-2, knottin-Fc, integrin binding-protein Fc fusion) aremade in transformed host cells using recombinant DNA techniques. To doso, a recombinant DNA molecule coding for the peptide is prepared.Methods of preparing such DNA molecules are well known in the art. Forinstance, sequences coding for the peptides could be excised from DNAusing suitable restriction enzymes. Alternatively, the DNA moleculecould be synthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

The methods of making polypeptides also include a vector capable ofexpressing the peptides in an appropriate host. The vector comprises theDNA molecule that codes for the peptides operatively linked toappropriate expression control sequences. Methods of affecting thisoperative linking, either before or after the DNA molecule is insertedinto the vector, are well known. Expression control sequences includepromoters, activators, enhancers, operators, ribosomal nuclease domains,start signals, stop signals, cap signals, polyadenylation signals, andother signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3^(rd) ed.) 2: 257-527. Solid phase synthesis is the preferredtechnique of making individual peptides since it is the mostcost-effective method of making small peptides. Compounds that containderivatized peptides or which contain non-peptide groups may besynthesized by well-known organic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

1. Expression of Polypeptides

The nucleic acid molecules described above can be contained within avector that is capable of directing their expression in, for example, acell that has been transduced with the vector. Accordingly, in additionto extended-PK IL-2 and knottin-Fc mutants, expression vectorscontaining a nucleic acid molecule encoding an extended-PK IL-2 orknottin-Fc mutant and cells transfected with these vectors are among thecertain embodiments.

Vectors suitable for use include T7-based vectors for use in bacteria(see, for example, Rosenberg et al., Gene 56: 125, 1987), the pMSXNDexpression vector for use in mammalian cells (Lee and Nathans, J. Biol.Chem. 263:3521, 1988), and baculovirus-derived vectors (for example theexpression vector pBacPAKS from Clontech, Palo Alto, Calif.) for use ininsect cells. The nucleic acid inserts, which encode the polypeptide ofinterest in such vectors, can be operably linked to a promoter, which isselected based on, for example, the cell type in which expression issought. For example, a T7 promoter can be used in bacteria, a polyhedrinpromoter can be used in insect cells, and a cytomegalovirus ormetallothionein promoter can be used in mammalian cells. Also, in thecase of higher eukaryotes, tissue-specific and cell type-specificpromoters are widely available. These promoters are so named for theirability to direct expression of a nucleic acid molecule in a giventissue or cell type within the body. Skilled artisans are well aware ofnumerous promoters and other regulatory elements which can be used todirect expression of nucleic acids.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neo^(r)) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Those of skill in the art can readily determinewhether a given regulatory element or selectable marker is suitable foruse in a particular experimental context.

Viral vectors that can be used in the invention include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells that contain and express a nucleic acidmolecule that encodes an extended-PK IL-2 or an integrin binding-proteinFc fusion mutant are also features of the invention. A cell of theinvention is a transfected cell, i.e., a cell into which a nucleic acidmolecule, for example a nucleic acid molecule encoding an extended-PKIL-2 mutant or integrin binding-protein Fc fusion, has been introducedby means of recombinant DNA techniques. The progeny of such a cell arealso considered within the scope of the invention.

The precise components of the expression system are not critical. Forexample, an extended-PK IL-2 or integrin binding-protein Fc fusionmutant can be produced in a prokaryotic host, such as the bacterium E.coli, or in a eukaryotic host, such as an insect cell (e.g., an Sf21cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLacells). These cells are available from many sources, including theAmerican Type Culture Collection (Manassas, Va.). In selecting anexpression system, it matters only that the components are compatiblewith one another. Artisans or ordinary skill are able to make such adetermination. Furthermore, if guidance is required in selecting anexpression system, skilled artisans may consult Ausubel et al. (CurrentProtocols in Molecular Biology, John Wiley and Sons, New York, N.Y.,1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985Suppl. 1987).

The expressed polypeptides can be purified from the expression systemusing routine biochemical procedures, and can be used, e.g., astherapeutic agents, as described herein.

XVI. Pharmaceutical Compositions and Modes of Administration

In some embodiments, the integrin-binding polypeptide-Fc fusion isadministered alone. In some embodiments, IL-2 is administered together(simultaneously or sequentially) with an integrin-binding polypeptide-Fcfusion. In some embodiments, IL-2 is administered prior to theadministration of an integrin-binding polypeptide-Fc fusion. In someembodiments, IL-2 is administered concurrently with the administrationof an integrin-binding polypeptide-Fc fusion. In some embodiments, IL-2is administered subsequent to the administration of an integrin-bindingpolypeptide-Fc fusion. In some embodiments, the IL-2 and anintegrin-binding polypeptide-Fc fusion are administered simultaneously.In other embodiments, the IL-2 and an integrin-binding polypeptide-Fcfusion are administered sequentially. In some embodiments, the IL-2 andan integrin-binding polypeptide-Fc fusion are administered within one,two, or three days of administration of the other. In some embodiments,the IL-2 is administered at day 2, day 3, and/or day 4 beforeadministration of the integrin-binding polypeptide-Fc fusion. In someembodiments, the IL-2 is administered at day 2, day 3, and/or day 4after administration of the integrin-binding polypeptide-Fc fusion. Insome embodiments, the IL-2 is administered at day 2 after administrationof the integrin-binding polypeptide-Fc fusion. In some embodiments, theIL-2 is administered at day 3 after administration of theintegrin-binding polypeptide-Fc fusion. In some embodiments, the IL-2 isadministered at day 4 after administration of the integrin-bindingpolypeptide-Fc fusion.

While not being bound by theory, the hypothesized therapeutic mechanismof action for IL-2 in combination with an Fc-containing tumor-targetingmoiety such as an antibody or integrin-binding polypeptide-Fc fusion isto activate and amplify the CD8+ T cell response following treatmentwith the integrin-binding polypeptide-Fc fusion (Cancer Cell. 2015 Apr.13; 27(4):489-501.). IL-2 is well known to cause numerous significantclinical toxicities, and so it is desirable to minimize the dose andfrequency of IL-2 administration. Consequently, clinical protocolsinvolving subcutaneous administration of low dose IL-2 have been testedand found to be much better tolerated albeit with somewhat reducedefficacy (Journal of Clinical Oncology, Vol 21, No 16 (August 15), 2003:pp 3127-3132). This lower dose may be tested in mice by using a suitableallometric scaling algorithm for administration of Proleukin(http://www.fda.gov/downloads/Drugs/ . . . /Guidances/UCM078932.pdf),leading to a dose of about 4 micrograms administered subcutaneously. Inlieu of extended-PK IL-2, we show that it is possible to administer abetter-tolerated schedule of subcutaneous low dose IL-2 on a daily basisstarting one day after administration of an integrin-bindingpolypeptide-Fc fusion. Low dose IL-2 preferentially stimulates cellsexpressing the high affinity IL-2 receptor subunit alpha, also known asCD25. Immunosuppressive regulatory CD4+ T cells (Tregs) are known toexpress CD25 at a high level, and activated cytolytic CD8+ T cells(CTLs) also transiently express CD25. In an attempt to preferentiallystimulate Tregs, chronic subcutaneous low dose IL-2 has been testedsuccessfully in patients with graft versus host disease (ScienceTranslational Medicine Vol 5 Issue 179 179ra43). One skilled in the artmight therefore expect this immunosuppressive effect mediated by Tregsto interfere with the desired activating CTL response to subcutaneousIL-2. However, we do in fact find that combining an integrin-bindingpolypeptide-Fc fusion with daily subcutaneous low-dose IL-2 for threedays leads to significant therapeutic effects in animal models of cancer(see, FIG. 4 and FIG. 7).

In some embodiments, integrin-binding polypeptide-Fc fusion and IL-2 areadministered with an immune checkpoint inhibitor. In some embodimentsthe immune checkpoint inhibitor is an anti-PD-1 antibody. In someembodiments, the anti PD-1 antibody can include but is not limited tonivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab(lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibodyJS001 (ShangHai JunShi), monocloanl anti-PD-1 antibody TSR-042 (Tesaro,Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1monoclonal antibody BGB-A317 (BeiGene), and/or anti-PD-1 antibodySHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810(Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb),and/or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In someembodiments, the PD-1 antibody is from clone: RMP1-14 (rat IgG)—BioXcellcat #BP0146.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4antibody, such as for example ipilimumab (Yervoy, Bristol-Myers Squibb).In some embodiments, an antagonist of VEGF is used in place of an immunecheckpoint inhibitor.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. Agents include,but are not limited to, in vitro synthetically prepared chemicalcompositions, antibodies, antigen binding regions, and combinations andconjugates thereof. In certain embodiments, an agent can act as anagonist, antagonist, allosteric modulator, or toxin.

In some embodiments, the invention provides for separate pharmaceuticalcompositions comprising extended-PK IL-2 with a pharmaceuticallyacceptable diluent, carrier, solubilizer, emulsifier, preservativeand/or adjuvant, and another pharmaceutical composition comprising aintegrin-binding polypeptide-Fc fusion with a pharmaceuticallyacceptable diluent, carrier, solubilizer, emulsifier, preservativeand/or adjuvant. In certain embodiments, the invention further providesfor a separate pharmaceutical composition comprising an immunecheckpoint inhibitor (or an antagonist of VEGF) with a pharmaceuticallyacceptable diluent, carrier, solubilizer, emulsifier, preservativeand/or adjuvant. In certain embodiments, the pharmaceutical compositionscomprise both IL-2 or extended-PK IL-2 and integrin-bindingpolypeptide-Fc fusion with a pharmaceutically acceptable diluents,carrier, solubilizer, emulsifier, preservative and/or adjuvant. Incertain embodiments, the pharmaceutical composition comprises IL-2 orextended-PK IL-2, integrin-binding polypeptide-Fc fusion, and an immunecheckpoint modulator, including inhibitors and/or stimulators (or anantagonist of VEGF) with a pharmaceutically acceptable diluents,carrier, solubilizer, emulsifier, preservative and/or adjuvant.

In some embodiments, the invention provides for pharmaceuticalcompositions comprising 11-2 or extended-PK IL-2, together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant, and another pharmaceutical compositioncomprises an integrin-binding polypeptide-Fc fusion, together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant. In certain embodiments, the inventionprovides for pharmaceutical compositions comprising an immune checkpointinhibitor, together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant. In certainembodiments, each of the agents, e.g., IL-2, extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), can be formulated asseparate compositions. In some embodiments, acceptable formulationmaterials preferably are nontoxic to recipients at the dosages andconcentrations employed. In certain embodiments, the formulationmaterial(s) are for s.c. and/or I.V. administration. In certainembodiments, the pharmaceutical composition can contain formulationmaterials for modifying, maintaining or preserving, for example, the pH,osmolality, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration ofthe composition. In certain embodiments, suitable formulation materialsinclude, but are not limited to, amino acids (such as glycine,glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants(such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates orother organic acids); bulking agents (such as mannitol or glycine);chelating agents (such as ethylenediamine tetraacetic acid (EDTA));complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides; disaccharides; and other carbohydrates (such asglucose, mannose or dextrins); proteins (such as serum albumin, gelatinor immunoglobulins); coloring, flavoring and diluting agents;emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone);low molecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, PEG, sorbitan esters,polysorbates such as polysorbate 20, polysorbate 80, triton,tromethamine, lecithin, cholesterol, tyloxapal); stability enhancingagents (such as sucrose or sorbitol); tonicity enhancing agents (such asalkali metal halides, preferably sodium or potassium chloride, mannitolsorbitol); delivery vehicles; diluents; excipients and/or pharmaceuticaladjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company (1995). In certain embodiments,the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or10 mM NAOAC, pH 5.2, 9% Sucrose. In certain embodiments, the optimalpharmaceutical composition will be determined by one skilled in the artdepending upon, for example, the intended route of administration,delivery format and desired dosage. See, for example, Remington'sPharmaceutical Sciences, supra. In certain embodiments, suchcompositions may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of extended-PK IL-2, aknottin-Fc, and optionally an immune checkpoint inhibitor (or anantagonist of VEGF).

In some embodiments, the primary vehicle or carrier in a pharmaceuticalcomposition can be either aqueous or non-aqueous in nature. For example,in certain embodiments, a suitable vehicle or carrier can be water forinjection, physiological saline solution or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. In certain embodiments, the salinecomprises isotonic phosphate-buffered saline. In certain embodiments,neutral buffered saline or saline mixed with serum albumin are furtherexemplary vehicles. In certain embodiments, pharmaceutical compositionscomprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH4.0-5.5, which can further include sorbitol or a suitable substitutetherefore. In some embodiments, a composition comprising IL-2 orextended-PK IL-2, an integrin-binding polypeptide-Fc fusion, andoptionally an immune checkpoint inhibitor (or an antagonist of VEGF),can be prepared for storage by mixing the selected composition havingthe desired degree of purity with optional formulation agents(Remington's Pharmaceutical Sciences, supra) in the form of alyophilized cake or an aqueous solution. Further, in certainembodiments, a composition comprising IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), can be formulated as alyophilizate using appropriate excipients such as sucrose.

In some embodiments, the pharmaceutical composition can be selected forparenteral delivery. In some embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In some embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Insome embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desiredextended-PK IL-2, a knottin-Fc, and optionally an immune checkpointinhibitor (or an antagonist of VEGF), in a pharmaceutically acceptablevehicle. In certain embodiments, a vehicle for parenteral injection issterile distilled water in which IL-2 or extended-PK IL-2,integrin-binding polypeptide-Fc fusion and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), are formulated as asterile, isotonic solution, and properly preserved. In some embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In some embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In some embodiments, a pharmaceutical composition can be formulated forinhalation. In some embodiments, IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), can be formulated as adry powder for inhalation. In some embodiments, an inhalation solutioncomprising IL-2 or extended-PK IL-2, an integrin-binding polypeptide-Fcfusion, and optionally an immune checkpoint inhibitor (or an antagonistof VEGF), can be formulated with a propellant for aerosol delivery. Incertain embodiments, solutions can be nebulized. Pulmonaryadministration is further described in PCT application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, IL-2 or extended-PK IL-2,an integrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), that is administered inthis fashion can be formulated with or without those carrierscustomarily used in the compounding of solid dosage forms such astablets and capsules. In some embodiments, a capsule can be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In some embodiments, at least oneadditional agent can be included to facilitate absorption of IL-2 orextended-PK IL-2, an integrin-binding polypeptide-Fc fusion, andoptionally an immune checkpoint inhibitor (or an antagonist of VEGF). Insome embodiments, diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders can also be employed.

In some embodiments, a pharmaceutical composition can involve aneffective quantity of IL-2 extended-PK IL-2, an integrin-bindingpolypeptide-Fc fusion, and optionally an immune checkpoint inhibitor (oran antagonist of VEGF), in a mixture with non-toxic excipients which aresuitable for the manufacture of tablets. In some embodiments, bydissolving the tablets in sterile water, or another appropriate vehicle,solutions can be prepared in unit-dose form. In some embodiments,suitable excipients include, but are not limited to, inert diluents,such as calcium carbonate, sodium carbonate or bicarbonate, lactose, orcalcium phosphate; or binding agents, such as starch, gelatin, oracacia; or lubricating agents such as magnesium stearate, stearic acid,or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving IL-2 or extended-PK IL-2,an integrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), in sustained- orcontrolled-delivery formulations. In some embodiments, techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, PCT Application No. PCT/US93/00829 which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions, incorporated by reference herein. Insome embodiments, sustained-release preparations can includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481, incorporated by reference herein), copolymers of L-glutamicacid and gamma efhyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556(1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105(1982)), ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain embodiments,sustained release compositions can also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In some embodiments,where the composition is lyophilized, sterilization using this methodcan be conducted either prior to or following lyophilization andreconstitution. In certain embodiments, the composition for parenteraladministration can be stored in lyophilized form or in a solution. Insome embodiments, parenteral compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In some embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. In someembodiments, such formulations can be stored either in a ready-to-useform or in a form (e.g., lyophilized) that is reconstituted prior toadministration.

In some embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In some embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In some embodiments, the effective amount of a pharmaceuticalcomposition comprising extended-PK IL-2 and/or one or morepharmaceutical compositions comprising a knottin-Fc, and optionally animmune checkpoint inhibitor (or an antagonist of VEGF), to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), are being used, theroute of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient. In some embodiments, the clinician can titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. In certain embodiments, a typical dosage of IL-2 or extended-PKIL-2 and an integrin-binding polypeptide-Fc fusion can each range fromabout 0.1 μg/kg to up to about 100 mg/kg or more, depending on thefactors mentioned above. In certain embodiments, the dosage can rangefrom 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg;or 5 μg/kg up to about 100 mg/kg. In some embodiments, the dosage of anintegrin-binding polypeptide-Fc fusion can range from about 5 mg/kg toabout 50 mg/kg. In some embodiments, the dosage can range from about 10mg/kg to about 40 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10mg/kg to about 25 mg/kg, about 5 mg/kg to about 20 mg/kg, about 5 mg/kgto about 15 mg/kg, or about 5 mg/kg to about 10 mg/kg. In someembodiments, the dosage is about 10 mg/kg.

IL-2 dosages can include but are not limited to high doses (HD): 0.72MIU/kg every 8 hr×15 (80 MIU/m2/d); low dosages (LD): 8 MIU/m2/d; andsubcutaneous dosages (SC): 250,000 U/kg/dose (9.25 MIU/m2/d, 5 days perweek, dose halved in weeks 2-6). MIU refers to million internationalunits. Subcutaneous IL-2 has been shown to be well tolerated and toexhibit a 10% response rate to subcutaneous IL-2 (see, for example, JClin Oncol 21:3127-3132, 2003). As such, Other subcutaneous IL-2 dosagescan include 1 MIU/m2 d 2-7, 12-21; 12 MIU/m2 d 9-11 & 1-3 subsequentcycles (see, for example, Mani et al., Breast Cancer Research andTreatment, 117(1), 83-89. 2009) and 8.8 MIU/m2/d, 6.25 MIU/m2/d (14 MIUs.c. thrice weekly during weeks 2 to 5 and 10 MIU s.c. thrice weeklyduring weeks 6 to 9; see, for example, Clinical Cancer Research 12(23),7046-7053, 2006; see Tables 3-5 copied below). While the examplesprovided herein are directed to mice studies, such studies can betranslated to human patients, including the IL-2 dosing. For example, 9MIU/m2 in humans is equivalent to 3.6 μg in mice. The FDA humanequivalent dose (HED) based on body area (see, for example,http://www.fda.gov/downloads/drugs/guidances/ucm078932.pdf; incorporatedby reference herein; see tables 1 and 3 copied below). For example, 9MIU/m2/(37 kg/m2)=0.24 MIU/kg×12.3 (from table 3)=2.95 MIU/kg in miceand 2.95 MIU/kg/(16.4 MIU/mg)*0.02 kg/mouse=3.6 μg in mice.

TABLE 3 Conversion of Animal Doses to Human To Convert Animal To ConvertAnimal Dose in Dose in mg/kg to mg/kg to HED^(a) in mg/kg, Either. Dosein mg/m², Divide Multiply Species Multiply by k_(m) Animal Dose ByAnimal Dose By Human 37 — — Child 25 — — (20 kg)^(b) Mouse 3 12.3 0.08Hamster 5 7.4 0.13 Rat 6 6.2 0.16 Ferret 7 5.3 0.19 Guinea pig 8 4.60.22 Rabbit 12 3.1 0.32 Dog 20 1.8 0.54 Primates: Monkeys^(c) 12 3.10.32 Marmoset 6 6.2 0.16 Squirrel 7 5.3 0.19 monkey Baboon 20 1.8 0.54Micro-pig 27 1.4 0.73 Mini-pig 35 1.1 0.95 ^(a)Assumes 60 kg human. Forspecies not listed or for weights outside the standard ranges, HED canbe calculated from the following formula: HED = animal dose in mg/kg ×(animal weight in kg human weight in kg)^(0.33). ^(b)This k_(m) value ispresided for reference only since healthy children will rarely bevolunteers for phase 1 trials. ^(c)For example, cynomolgus, rhesus, andstumptail.

TABLE 4 Conversion of Animal Doses to Human Equivalent Doses Based onBody Surface Area To Convert Animal Dose in To Convert mg/kg to HED^(a)Reference Working Body Animal Dose in mg/kg, Either. Body Weight Surfacein mg/kg to Divide Multiply Weight Range^(a) Area Dose in mg/m², AnimalAnimal Species (kg) (kg) (m²) Multiply by k_(m) Dose By Dose By Human 60— 1.62 37 — — Child^(c) 20 — 0.80 25 — — Mouse 0.020 0.011-0.034 0.007 312.3 0.081 Hamster 0.080 0.047-0.157 0.016 5 7.4 0.135 Rat 0.1500.080-0.270 0.025 6 6.2 0.162 Ferret 0.300 0.160-0.540 0.043 7 5.3 0.189Guinea pig 0.400 0.208-0.700 0.05 8 4.6 0.216 Rabbit 1.8 0.9-3.0 0.15 123.1 0.324 Dog 1.0 5.17 0.50 20 1.8 0.541 Primates Monkey s^(d) 3 1.4-4.90.25 12 3.1 0.324 Marmoset 0.350 0.140-0.720 0.06 6 6.2 0.162 Squirrelmonkey 0.600 0.290-0.970 0.09 7 5.3 0.189 Baboon 12  7-23 0.60 20 1.80.541 Micro-pig 20 10-33 0.74 27 1.4 0.730 Mini-pig 40 25-64 1.14 35 1.10.946 ^(a)For animal weights within the specified ranges, the HED for a60 kg human calculated using the standard k_(m) value will not vary morethan ±20 percent from the HED calculated using a k_(m) value based onthe exact animal weight. ^(b) Assumes 60 kg human. For species notlisted or for weights outside the standaid ranges, HED can be calculatedfrom the following formula: HED = animal dose in mg/kg × (animal weightin kg human weight in kg)^(0.33). ^(c)This k_(m) value is presided forreference only since healthy children will rarely be volunteers forphase 1 trials. ^(d)For example, cynomolgus, rhesus, and stumptail.

Subcutaneous IL-2 has a plasma peak when 0.7 nM per MIU/m2 SC is dosed.The calculation is as follows:

-   -   10.6 CU/mL per MCU/m2;    -   10.6 CU/mL/(4×10{circumflex over ( )}6 CU/mg)*1000 mL/L/(15,300        mg/mmol)×10{circumflex over ( )}6 nmol/mmol=0.17 nM    -   16.4 MIU/mg/(4 MCU/mg)=4.1 MIU/MCU        This has been described in, for example, Cancer Research 50.        2009-2017, '90, Table 4 of which is copied below.

TABLE 5 Peak serum levels after s.c. injections Last time Dose levelpoint Peak time Peak level Peak Patient (MU/m²) (min) (min) (units/ml)level/dose 1 0.5 1440 120 6.7 13.4 2 0.5 1440 240 4.5 9.0 3 0.5 1440 3607.5 15.0 4 1.0 1440 120 5.7 5.7 5 1.0 1440 120 11.8 11.8 7 1.0 14.40 2409.5 9.5 Median 0.8 1440 180 7.1 10.7 Minimum 0.5 1440 120 4.5 5.7Maximum 1.0 1440 360 11.8 15.0 Mean ± SD 10.6 ± 3.5 IL-2 wasadministered by s.c. injection at doses of 0.5 or 1.0 MU/m², and theactivity was determined in serum taken at 0.5, 1, 2, 3, 4, 6, 8, and 24h after injection. The median dose normalized peak level of 10.7units/ml and time to peak of 180 min are similar to the correspondingvalues of 14.0 units/ml and 150 min observed after i.m. administration(Table 4). The dose levels were lower in this study as compared to thei.m. trial, and meaningful values for AUC could not be obtained.

With regard to Proleukin, it has a MW 15.3 kD and can be dosed at 16.4MIU/mg. Other dosage units have been described, including “Cetus Units”and “Roche Units”, and 1 Cetus Unit=3-6 IU (see, for example,http://cancerguide.org/rcc_i12.html Cancer Research 50. 2009-2017, '90).

With regard to surface area dosage conversion factors in IL-2 dosing,the following is applicable to the methods described herein. In adulthumans, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq.m.=3700 mg/m2. Agiven mg/kg dose in mice can be divided by 12 to give an equivalent dosein man in terms of mg/m2. For example, a 60 kg human has 1.6 m2 surfacearea. See, for example,https://ncifrederick.cancer.gov/Lasp/Acuc/Frederick/Media/Documents/ACUC42.pdf

In some embodiments, a typical dosage for an immune checkpoint inhibitorcan range from about 0.1 mg/kg to up to about 300 mg/kg or more,depending on the factors mentioned above. In some embodiments, thedosage can range from 1 mg/kg up to about 300 mg/kg; or 5 mg/kg up toabout 300 mg/kg; or 10 mg/kg up to about 300 mg/kg.

In some embodiments, a typical dosage for an immune checkpointstimulator can range from about 0.1 mg/kg to up to about 300 mg/kg ormore, depending on the factors mentioned above. In some embodiments, thedosage can range from 1 mg/kg up to about 300 mg/kg; or 5 mg/kg up toabout 300 mg/kg; or 10 mg/kg up to about 300 mg/kg.

In some embodiments, a typical dosage for IFNα can range from about 1-30million Units/m², depending on the factors mentioned above. In someembodiments, the dosage can range from about 2-25 million Units/m². Insome embodiments, the dosage can range from about 2-20 million Units/m².In some embodiments, the dosage can range from about 5-20 millionUnits/m². In some embodiments, the dosage can range from about 10-20million Units/m². In some embodiments, the dosage can range from about5-15 million Units/m². In some embodiments, the dosage can range fromabout 15-20 million Units/m². In some embodiments, the dosage can rangefrom about 5-10 million Units/m². In some embodiments, the IFN-α isIntron-A, commercially available from Merck (see, for example, U.S. Pat.No. 6,610,830 andhttps://www.merck.com/product/usa/pi_circulars/i/intron_a/intron_a_pi.pdp.In some embodiments, the IFN-α is PEG-IFN-α. In some embodiments, theIFN-α is Pegintron (see, for example, U.S. Pat. Nos. 6,610,830 and6,180,096). In some embodiments, the IFN-α is SYLATRON (see, forexample, U.S. Pat. Nos. 6,610,830 and 6,180,096). In some embodiments,PEG-IFNα can be administered at about 0.25-2.5 μg/kg, or about 0.5-1.5μg/kg (see, for example, U.S. Pat. No. 6,524,570). In some embodiments,PEG-IFNα can be administered at about 0.25-2.5 μg/kg. In someembodiments, PEG-IFNα can be administered at about 0.5-2.5 μg/kg. Insome embodiments, PEG-IFNα can be administered at about 1-2.5 μg/kg. Insome embodiments, PEG-IFNα can be administered at about 1.5-2.5 μg/kg.In some embodiments, PEG-IFNα can be administered at about 0.5-1.5μg/kg. In some embodiments, PEG-IFNα can be administered at about 0.5-1μg/kg. In some embodiments, PEG-IFNα can be administered at about 1-1.5μg/kg.

In some embodiments, the frequency of dosing will take into account thepharmacokinetic parameters of IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), in the formulationused. In some embodiments, a clinician will administer the compositionuntil a dosage is reached that achieves the desired effect. In someembodiments, the composition can therefore be administered as a singledose, or as two or more doses (which may or may not contain the sameamount of the desired molecule) over time, or as a continuous infusionvia an implantation device or catheter. Further refinement of theappropriate dosage can be made by those of ordinary skill in the art andis within the ambit of tasks routinely performed by them. In someembodiments, appropriate dosages can be ascertained through use ofappropriate dose-response data. In some embodiments, IL-2 isadministered before, after, and/or simultaneously with theintegrin-binding polypeptide-Fc fusion. In some embodiments, IL-2 isadministered 1 day, 2 days, 3 days, 4 days, 5, days, 6 days, or moreafter administration of the integrin-binding polypeptide-Fc fusion. Insome embodiments, IL-2 is administered 2 days after administration ofthe integrin-binding polypeptide-Fc fusion. In some embodiments, IL-2 isadministered 3 days after administration of the integrin-bindingpolypeptide-Fc fusion. In some embodiments, IL-2 is administered 4 daysafter administration of the integrin-binding polypeptide-Fc fusion.

In some embodiments, the IL-2 is administered at a 12 MIU/m2 or lowerdaily dose. In some embodiments, the IL-2 dose is less than 14 MIU/m2,less than 12 MIU/m2, less than 10 MIU/m2, less than 8 MIU/m2, less than6 MIU/m2, less than 4 MIU/m2, less than 2 MIU/m2 per day. In someembodiments, the IL-2 dose is about 14 MIU/m2 to about 6 MIU/m2 per day.In some embodiments, the IL-2 dose is about 12 MIU/m2 to about 8 MIU/m2per day. In some embodiments, the IL-2 dose is about 12 MIU/m2 to about10 MIU/m2 per day.

In some embodiments, the route of administration of the pharmaceuticalcomposition is in accord with known methods, e.g. orally, throughinjection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In some embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.In certain embodiments, individual elements of the combination therapymay be administered by different routes.

In some embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In someembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration. In some embodiments, it can be desirable to use apharmaceutical composition comprising IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint inhibitor (or an antagonist of VEGF), in an ex vivo manner.In such instances, cells, tissues and/or organs that have been removedfrom the patient are exposed to a pharmaceutical composition comprisingIL-2 or extended-PK IL-2, an integrin-binding polypeptide-Fc fusion, andoptionally an immune checkpoint inhibitor (or an antagonist of VEGF),after which the cells, tissues and/or organs are subsequently implantedback into the patient.

In some embodiments, IL-2 or extended-PK IL-2, an integrin-bindingpolypeptide-Fc fusion, and optionally an immune stiumulator or immunecheckpoint inhibitor (or an antagonist of VEGF), can be delivered byimplanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides. In certain embodiments, such cells can be animal or humancells, and can be autologous, heterologous, or xenogeneic. In someembodiments, the cells can be immortalized. In some embodiments, inorder to decrease the chance of an immunological response, the cells canbe encapsulated to avoid infiltration of surrounding tissues. In someembodiments, the encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

XVII. Methods of Treatment & Therapeutic Efficacy Readouts

The integrin-binding polypeptide-Fc fusions and/or nucleic acidsexpressing them, as described herein, are useful for treating a disorderassociated with abnormal apoptosis or a differentiative process (e.g.,cellular proliferative disorders or cellular differentiative disorders,such as cancer). Additionally, the IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunecheckpoint modulator, including an inhibitor or stimulator (or anantagonist of VEGF), and/or nucleic acids expressing them, as describedherein, are useful for treating a disorder associated with abnormalapoptosis or a differentiative process (e.g., cellular proliferativedisorders or cellular differentiative disorders, such as cancer).Non-limiting examples of cancers that are amenable to treatment with themethods of the present invention are described below.

Examples of cellular proliferative and/or differentiative disordersinclude cancer (e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver.Accordingly, the compositions used herein, comprising, e.g., extended-PKIL-2, a knottin-Fc, and optionally an immune checkpoint inhibitor (or anantagonist of VEGF), can be administered to a patient who has cancer.

As used herein, we may use the terms “cancer” (or “cancerous”),“hyperproliferative,” and “neoplastic” to refer to cells having thecapacity for autonomous growth (i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth).

Hyperproliferative and neoplastic disease states may be categorized aspathologic (i.e., characterizing or constituting a disease state), orthey may be categorized as non-pathologic (i.e., as a deviation fromnormal but not associated with a disease state). The terms are meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness.“Pathologic hyperproliferative” cells occur in disease statescharacterized by malignant tumor growth. Examples of non-pathologichyperproliferative cells include proliferation of cells associated withwound repair.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. In some embodiments, the diseasesarise from poorly differentiated acute leukemias (e.g., erythroblasticleukemia and acute megakaryoblastic leukemia). Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macro globulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The mutant IL-2 polypeptidescan be used to treat patients who have, who are suspected of having, orwho may be at high risk for developing any type of cancer, includingrenal carcinoma or melanoma, or any viral disease. Exemplary carcinomasinclude those forming from tissue of the cervix, lung, prostate, breast,head and neck, colon and ovary. The term also includes carcinosarcomas,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive non-invasive or metastatic) characterized by abnormaland uncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth). Non-limiting examples of which are describedherein. This includes any physiological condition in mammals that istypically characterized by unregulated cell growth. Examples of cancerare exemplified in the working examples and also are described withinthe specification. The terms “cancer” or “neoplasm” are used to refer tomalignancies of the various organ systems, including those affecting thelung, breast, thyroid, lymph glands and lymphoid tissue,gastrointestinal organs, and the genitourinary tract, as well as toadenocarcinomas which are generally considered to include malignanciessuch as most colon cancers, renal-cell carcinoma, prostate cancer and/ortesticular tumors, non-small cell carcinoma of the lung, cancer of thesmall intestine and cancer of the esophagus.

Non-limiting examples of cancers that can be treated using theintegrin-binding polypeptide-Fc fusions of the invention include, butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, lung cancer (including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, and squamous carcinoma of thelung), cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer (including gastrointestinal cancer), pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer, aswell as B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; multiple myeloma andpost-transplant lymphoproliferative disorder (PTLD). In someembodiments, other cancers amenable for treatment by the presentinvention include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include colorectal, bladder,ovarian, melanoma, squamous cell cancer, lung cancer (includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.Preferably, the cancer is selected from the group consisting ofcolorectal cancer, breast cancer, rectal cancer, non-small cell lungcancer, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostatecancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi'ssarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovariancancer, mesothelioma, and multiple myeloma. In an exemplary embodimentthe cancer is an early or advanced (including metastatic) bladder,ovarian or melanoma. In another embodiment the cancer is colorectalcancer. In some embodiments, the methods of the present invention areuseful for the treatment of vascularized tumors.

It will be appreciated by those skilled in the art that amounts for eachof the IL-2, extended-PK IL-2, IFNα, integrin-binding polypeptide-Fcfusion, and optionally an immune checkpoint modulator, including aninhibitor or stimulator (or an antagonist of VEGF), that are sufficientto reduce tumor growth and size, or a therapeutically effective amount,will vary not only on the particular compounds or compositions selected,but also with the route of administration, the nature of the conditionbeing treated, and the age and condition of the patient, and willultimately be at the discretion of the patient's physician orpharmacist. The length of time during which the compounds used in theinstant method will be given varies on an individual basis. As describedherein, immune checkpoint inhibitors include anti-PD-1 antibodies,anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and immune stimulatorsinclude anti-4-1BB/CD137 antibodies.

It will be appreciated by those skilled in the art that the coloncarcinoma model used herein in the examples (MC38 murine coloncarcinoma) is a generalized model for solid tumors. That is, efficacy oftreatments in this model is also predictive of efficacy of thetreatments in other non-melanoma solid tumors. For example, as describedin Baird et al. (J Immunology 2013; 190:469-78; Epub Dec. 7, 2012),efficacy of cps, a parasite strain that induces an adaptive immuneresponse, in mediating anti-tumor immunity against B16F10 tumors wasfound to be generalizable to other solid tumors, including models oflung carcinoma and ovarian cancer. In another example, results from aline of research into VEGF targeting lymphocytes also shows that resultsin B16F10 tumors were generalizable to the other tumor types studied(Chinnasamy et al., JCI 2010; 120:3953-68; Chinnasamy et al, Clin CancerRes 2012; 18: 1672-83). In yet another example, immunotherapy involvingLAG-3 and PD-1 led to reduced tumor burden, with generalizable resultsin a fibrosarcoma and colon adenocarcinoma cell lines (Woo et al.,Cancer Res 2012; 72:917-27).

In some embodiments, the integrin-binding polypeptide-Fc fusions areused to treat cancer. In some embodiments, the integrin-bindingpolypeptide-Fc fusions, and optional immune checkpoint stimulator orimmune checkpoint inhibitor (or an antagonist of VEGF), are used totreat cancer. In some embodiments, the IL-2 or extended-PK IL-2,integrin-binding polypeptide-Fc fusions, and optional immune stimulatoror immune checkpoint inhibitor (or an antagonist of VEGF), are used totreat cancer. In some embodiments, the IL-2, integrin-bindingpolypeptide-Fc fusions, and optional immune stimulator or immunecheckpoint inhibitor (or an antagonist of VEGF), are used to treatcancer. In some embodiments, the extended-PK IL-2, integrin-bindingpolypeptide-Fc fusions, and optional immune stimulator or immunecheckpoint inhibitor (or an antagonist of VEGF), are used to treatcancer.

In some embodiments, the IL-2 or extended-PK IL-2, IFNα,integrin-binding polypeptide-Fc fusions, and optional immune stimulatoror immune checkpoint inhibitor (or an antagonist of VEGF) are used totreat melanoma, leukemia, lung cancer, breast cancer, prostate cancer,ovarian cancer, colon cancer, renal carcinoma, and brain cancer.

In some embodiments, the IL-2 or extended-PK IL-2, IFNα,integrin-binding polypeptide-Fc fusions and optional immune stimulatoror immune checkpoint inhibitor (or an antagonist of VEGF) inhibit growthand/or proliferation of tumor cells.

In some embodiments, the IL-2 or extended-PK IL-2, IFNα,integrin-binding polypeptide-Fc fusions, and optional immune stimulatoror immune checkpoint inhibitor (or an antagonist of VEGF) reduce tumorsize.

In some embodiments, the IL-2 extended-PK IL-2, IFNα, integrin-bindingpolypeptide-Fc fusions, and optional immune stimulator or immunecheckpoint inhibitor (or an antagonist of VEGF) inhibit metastases of aprimary tumor.

In some embodiments, an integrin-binding polypeptide-Fc fusions and animmune stimulator or immune checkpoint inhibitor (or an antagonist ofVEGF), with or without IL-2, inhibit growth and/or proliferation oftumor cells. In some embodiments, an integrin-binding polypeptide-Fcfusions and an immune stimulator or immune checkpoint inhibitor (or anantagonist of VEGF), with or without IL-2, reduce tumor size. In certainembodiments, an integrin-binding polypeptide-Fc fusions and an immunestimulator or immune checkpoint inhibitor, with or without IL-2, inhibitmetastases of a primary tumor.

In some embodiments, an integrin-binding polypeptide-Fc fusions and animmune stimulator or immune checkpoint inhibitor (or an antagonist ofVEGF), with or without IFNα, inhibit growth and/or proliferation oftumor cells. In some embodiments, an integrin-binding polypeptide-Fcfusions and an immune stimulator or immune checkpoint inhibitor (or anantagonist of VEGF), with or without IFNα, reduce tumor size. In certainembodiments, an integrin-binding polypeptide-Fc fusions and an immunestimulator or immune checkpoint inhibitor, with or without IFNα, inhibitmetastases of a primary tumor.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment of thenoted cancers and symptoms.

“Cancer therapy” herein refers to any method which prevents or treatscancer or ameliorates one or more of the symptoms of cancer. Typically,such therapies will comprise administration of integrin-bindingpolypeptide-Fc fusions either alone or in combination with chemotherapyor radiotherapy or other biologics and for enhancing the activitythereof. In some embodiments, cancer therapy can include or be measuredby increased survival. In some embodiments, cancer therapy results in areduction in tumor volume.

In some embodiments, increased survival effects are observed withcombinations comprising NOD201+anti-CTLA-4, NOD201+anti-PD-L1 antibody,NOD201+anti-4-1BB/CD137 antibody, and NOD201+anti-PD-1 antibody ascompared to NOD201 alone. In some embodiments, the combination furthercomprises IL-2. In some embodiments, the combination further comprisesIFNα. In some embodiments, increased survival effects are observed withNOD201 in combination with anti-PD-1 antibody and IL-2. In someembodiments, increased survival effects are observed with NOD201 incombination with anti-PD-1 antibody and low dose IL-2.

In some embodiments, increased survival effects are not observed withcombinations comprising NOD201+anti-LAG-3 antibody, NOD201+anti-TIM-3antibody, or NOD201+anti-TIGIT antibody as compared to NOD201 alone.

In some embodiments, a reduction in tumor volume is observed withcombinations comprising NOD201+anti-CTLA-4, NOD201+anti-PD-L1 antibody,NOD201+anti-4-1BB/CD137 antibody, and NOD201+anti-PD-1 antibody ascompared to NOD201 alone. In some embodiments, the combination furthercomprises IL-2. In some embodiments, the combination further comprisesIFNα. In some embodiments, a reduction in tumor volume is observed withNOD201 in combination with anti-PD-1 antibody and IL-2. In someembodiments, a reduction in tumor volume is observed with NOD201 incombination with anti-PD-1 antibody and low dose IL-2.

In some embodiments, a reduction in tumor volume are not observed withcombinations NOD201+anti-LAG-3 antibody, NOD201+anti-TIM-3 antibody, orNOD201+anti-TIGIT antibody as compared to NOD201 alone.

In some embodiments, the patient can be examined for markers related todetermining treatment efficacy for treatment with NOD201 and ananti-PD-1 antibody. In some embodiments, these markers are T-cell genesubsets, including T-cell activation genes. (See, for example FIGS. 35,36, 37, 38, and 39). In some embodiments, there is a significantdifference in expression T-cell activation genes in therapy respondersversus non-responder (see, for example, FIG. 39). In some embodiments,there is a significant difference in expression T-cell activation genesin therapy responders versus non-responders for treatment with NOD201and an anti-PD-1 antibody. In some embodiments, there is a significantdifference in expression of Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2,Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Nck1, Rac3,Nck2, and/or PAK3 in therapy responders versus non-responders. In someembodiments, there is a significant difference in expression of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, Vav2, Cdc42, Nck1, Rac3, Nck2, and/or PAK3 in therapy respondersversus non-responders for treatment with NOD201 and an anti-PD-1antibody. In some embodiments, there is a significant difference inexpression of Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3, and/or Pak3 in therapyresponders versus non-responders. In some embodiments, there is asignificant difference in expression of Lat, Pak1, Vav3, Grap3, Grb2,Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3,and/or Pak3 in therapy responders versus non-responders for treatmentwith NOD201 and an anti-PD-1 antibody. In some embodiments, there is asignificant difference in expression of Lat, Pak1, Vav3, Grap3, Grb2,Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3,and/or Pak3 in therapy responders versus non-responders. In someembodiments, there is a significant difference in expression of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, Vav2, Cdc42, Rac3, and/or Pak3 in therapy responders versusnon-responders for treatment with NOD201 and an anti-PD-1 antibody. Insome embodiments, Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1,Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3, and/or Pak3 areupregulated in therapy responders versus non-responders. In someembodiments, Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3, and/or Pak3 are upregulatedin therapy responders versus non-responders for treatment with NOD201and an anti-PD-1 antibody. In some embodiments, Lat, Pak1, Vav3, Grap3,Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42,Rac3, and/or Pak3 are downregulated in therapy non-responders versusresponders. In some embodiments, Lat, Pak1, Vav3, Grap3, Grb2, Was,Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3,and/or Pak3 are downregulated in therapy non-responders versusresponders for treatment with NOD201 and an anti-PD-1 antibody. In someembodiments, Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, and/or Vav2 are upregulated in therapyresponders versus non-responders. In some embodiments, Lat, Pak1, Vav3,Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2,and/or Vav2 are upregulated in therapy responders versus non-respondersfor treatment with NOD201 and an anti-PD-1 antibody. In someembodiments, Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, and/or Vav2 are downregulated in therapynon-responders versus responders. In some embodiments, Lat, Pak1, Vav3,Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2,and/or Vav2 are downregulated in therapy non-responders versusresponders for treatment with NOD201 and an anti-PD-1 antibody. In someembodiments, there is a significant difference in expression of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, and/or Vav2 in therapy responders versus non-responders. In someembodiments, there is a significant difference in expression of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, and/or Vav2 in therapy responders versus non-responders fortreatment with NOD201 and an anti-PD-1 antibody. In some embodiments,upregulation of one of more of Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2,Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Nck1, Rac3,Nck2, and/or Pak3 is indicative of the subject being responsive totreatment. In some embodiments, upregulation of one of more of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, Vav2, Cdc42, Nck1, Rac3, Nck2, and/or Pak3 is indicative of thesubject being responsive to treatment with NOD201 and an anti-PD-1antibody. In some embodiments, there is a significant difference inexpression of Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Nck1, Rac3, Nck2, and/or PAK3 isindicative of the subject being responsive to treatment. In someembodiments, there is a significant difference in expression of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, Vav2, Cdc42, Nck1, Rac3, Nck2, and/or PAK3 is indicative of thesubject being responsive to treatment with NOD201 and an anti-PD-1antibody. In some embodiments, upregulation of one of more of Lat, Pak1,Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2,Vav2, Cdc42, Rac3, and/or Pak3 is indicative of the subject beingresponsive to treatment. In some embodiments, upregulation of one ofmore of Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3, and/or Pak3 is indicative ofthe subject being responsive to treatment with NOD201 and an anti-PD-1antibody. In some embodiments, downregulation of one of more of Lat,Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1,Sos2, Vav2, Cdc42, Nck1, Rac3, Nck2, and/or Pak3 is indicative of thesubject being non-responsive to treatment. In some embodiments,downregulation of one of more of Lat, Pak1, Vav3, Grap3, Grb2, Was,Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Nck1,Rac3, Nck2, and/or Pak3 is indicative of the subject beingnon-responsive to treatment with NOD201 and an anti-PD-1 antibody. Insome embodiments, there is a significant difference in expression ofLat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2,Sos1, Sos2, Vav2, Cdc42, Nck1, Rac3, Nck2, and/or PAK3 is indicative ofthe subject being non-responsive to treatment. In some embodiments,there is a significant difference in expression of Lat, Pak1, Vav3,Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2,Cdc42, Nck1, Rac3, Nck2, and/or PAK3 is indicative of the subject beingnon-responsive to treatment with NOD201 and an anti-PD-1 antibody. Insome embodiments, downregulation of one of more of Lat, Pak1, Vav3,Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1, Plcg1, Pak2, Sos1, Sos2, Vav2,Cdc42, Rac3, and/or Pak3 is indicative of the subject beingnon-responsive to treatment. In some embodiments, downregulation of oneof more of Lat, Pak1, Vav3, Grap3, Grb2, Was, Rac2, Lcp2, Vav1, Rac1,Plcg1, Pak2, Sos1, Sos2, Vav2, Cdc42, Rac3, and/or Pak3 is indicative ofthe subject being non-responsive to treatment with NOD201 and ananti-PD-1 antibody.

Efficacy readouts can include monitoring for changes in αβ and/or γδ Tcells, cytotoxic T cell activity, changes in markers such as CD137,CD107a, changes in NK and/or NK/T activity, interferon-γ production,changes in regulatory T-cell (including changes in Treg number), changesin macrophage number, changes in neutrophil pro-tumorigenic activity,T-cell activation, CTL activation, changes in activation markers such asCD45RA or CCR7, as well as cancer cell cytotoxicity assays. Efficacyreadouts can also include examination of expression of the genesprovided in FIGS. 35, 36, 37, 38, and 39, and as discussed above.Efficacy readouts can also include tumor size reduction, tumor numberreduction, reduction in the number of metastases, and decreased diseasestate (or increased life expectancy). In some embodiments, a reductionin tumor size by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or100% is indicative of therapeutic efficacy. In some embodiments, areduction in tumor number by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 100% is indicative of therapeutic efficacy. In someembodiments, a reduction in tumor burden by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 100% is indicative of therapeutic efficacy.In some embodiments, a reduction in the number of metastases by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% is indicative oftherapeutic efficacy.

XVIII. Kits

A kit can include an integrin-binding polypeptide-Fc fusion andoptionally an immune stimulator or immune checkpoint inhibitor (or anantagonist of VEGF), as disclosed herein, and instructions for use.Additionally, a kit can include IL-2 or extended-PK IL-2, anintegrin-binding polypeptide-Fc fusion, and optionally an immunestimulator or immune checkpoint inhibitor (or an antagonist of VEGF), asdisclosed herein, and instructions for use. The kits may comprise, in asuitable container, IL-2 or extended-PK IL-2, IFNα, an integrin-bindingpolypeptide-Fc fusion, an optional immune stimulator or immunecheckpoint inhibitor (or an antagonist of VEGF), one or more controls,and various buffers, reagents, enzymes and other standard ingredientswell known in the art. Some embodiments include a kit with extended-PKIL-2, IFNα, knottin-Fc, and optional immune stimulator or immunecheckpoint inhibitor (or an antagonist of VEGF) in the same vial. Incertain embodiments, a kit includes extended-PK IL-2, IFNα, knottin-Fc,and optional immune stimulator or immune checkpoint inhibitor (or anantagonist of VEGF) in separate vials.

The container can include at least one vial, well, test tube, flask,bottle, syringe, or other container means, into which IL-2 orextended-PK IL-2, IFNα, an integrin-binding polypeptide-Fc fusion, andoptionally an immune stimulator or immune checkpoint inhibitor (or anantagonist of VEGF) may be placed, and in some instances, suitablyaliquoted. Where an additional component is provided, the kit cancontain additional containers into which this component may be placed.The kits can also include a means for containing IL-2 or extended-PKIL-2, IFNα, an integrin-binding polypeptide-Fc fusion, and optionally animmune stimulator or immune checkpoint inhibitor (or an antagonist ofVEGF) and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained. Containersand/or kits can include labeling with instructions for use and/orwarnings.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference. In particular, the disclosures ofInternational Patent Publication No. WO 2013/177187, U.S. Pat. No.8,536,301, and U.S. Patent Publication No. 2014/0073518 are expresslyincorporated herein by reference in their entireties for all purposes.

EXAMPLES

Below are examples of specific embodiments for carrying out the methodsdescribed herein. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperatures, etc.), but some experimentalerror and deviation should, of course, be allowed for. The practice ofthe present invention will employ, unless otherwise indicated,conventional methods of protein chemistry, biochemistry, recombinant DNAtechniques and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., T. E.Creighton, Proteins: Structures and Molecular Properties (W.H. Freemanand Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,Inc., current addition); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2^(nd) Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18^(th) Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed.(Plenum Press) Vols A and B(1992).

Moreover, while the examples below employ IL-2 of mouse origin and mouseFc fused as part of the integrin-binding polypeptide-Fc fusion, itshould be understood that corresponding human IL-2 or extended-PK IL-2(i.e., human serum albumin (HSA) and human IL-2, and variants thereof)and integrin-binding polypeptide-Fc fusions comprising a human Fc (i.e.,Fc from human IgG1 fused to the integrin-binding polypeptide) can bereadily generated by those of ordinary skill in the art using methodsdescribed supra, and used in the methods disclosed herein.

Example 1 MC38-NODU-E202 Materials and Methods

Three potential therapeutic candidates were designed and tested. Thesevariants comprised our tumor targeting peptide (2.5F) fused to anantibody Fc domain (human IgG1). NOD201: no linker, NOD203: a short[Gly₄Ser] linker, and NOD204: a long [Gly4Ser3] linker. These threeconstructs were expressed with a signal peptide derived from theAzurocidin preproprotein (note: the signal peptide is a sequence thatdirects the protein expression within the cell). Genes encoding for theopen reading frames of these constructs were codon optimized formammalian cell expression, and protein constructs were produced bytransient expression in human embryonic kidney cells. 100 mL cultureswere purified by MabSelect SuRe resin, quantified by UV/Vis absorbance,and analyzed by SDS-PAGE and size exclusion chromatography. The proteinswere >98% monomer (i.e. lack of aggregates) when analyzed by sizeexclusion chromatography.

Serum stability of the proteins was measured after incubation in mouseserum for 48 hours at 37° C. as compared to untreated sample. Proteinintegrity was measured by binding to αvβ3 integrin by BiolayerInterferometry. All NOD fusion proteins were not compromised afterincubation in mouse serum.

Melting temperature (Tm) was measured in two different buffers, PBS andcitrate. Tm for all proteins was 68° C. in PBS and 66° C. in citrate.

NOD201X, which contains a scrambled integrin binding sequence, was alsocloned and produced in in HEK cells as a negative control.

NOD201M contains the 2.5F peptide fused to a mouse Fc domain. Thisconstruct is necessary for experiments in syngeneic mouse experiments(mice with an intact immune system). NOD201M was produced in HEK cellsfor animal experiments.

NOD201 was found to be a “Universal” tumor targeting agent, capable ofpotentiating T cell directed cancer immunotherapies (e.g. checkpointblockade, IL-2) as well as driving T cell infiltration of tumors throughtargeting innate effector functions to integrins.

While not being bound by theory, the proposed NOD201 mechanism of actionis that ADCC drives cross priming of T cell response. This is believedto require: Fc effector functions, macrophages, CD8+ T cells, and CD8+dendritic cells. Fc effector functions create inflammatory TME (tumormicroenvironment), which results in increased intratumoral chemokines.

Example 2 MC38-NODU-E202 Materials and Methods Mice

Female C57BL/6 mice (C57BL/6/NCrl, Charles River) were seven weeks oldon D1 of the study and had a BW range of 15.5-21.8 g. The animals werefed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified andIrradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat,and 5.0% crude fiber. The mice were housed on irradiated Enrich-o′cobs™bedding in static microisolators on a 12-hour light cycle at 20-22° C.(68-72° F.) and 40-60% humidity. CR Discovery Services specificallycomplies with the recommendations of the Guide for Care and Use ofLaboratory Animals with respect to restraint, husbandry, surgicalprocedures, feed and fluid regulation, and veterinary care. The animalcare and use program at CR Discovery Services is accredited by theAssociation for Assessment and Accreditation of Laboratory Animal CareInternational, which assures compliance with accepted standards for thecare and use of laboratory animals.

Tumor Cell Culture

MC38 murine colon carcinoma cells provided by Nodus Therapeutics, Inc.were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with10% fetal bovine serum and 2 mM glutamine, 100 units/mL penicillin Gsodium, 100 μg/mL streptomycin sulfate, and 25 μg/mL gentamicin. Cellcultures were maintained in tissue culture flasks in a humidifiedincubator at 37° C., in an atmosphere of 5% CO₂ and 95% air.

Tumor Implantation and Measurement

The MC38 colon cells used for implantation were harvested during logphase growth and resuspended in cold RPMI media. Mice were anesthetizedwith isoflurane prior to implantation. Each mouse was injectedsubcutaneously in the right flank with 1×10⁶ tumor cells (0.1 mL cellsuspension) and tumors were monitored as their volumes approached thetarget range of 60 to 180 mm³. Seven Days after tumor implantation, onD1 of the study, animals with individual tumor volumes ranging from 63to 172 mm³ were sorted into eleven groups (n=10) with group mean tumorvolumes ranging from 109-112 mm³. Tumors were measured with caliperstwice weekly in two dimensions. Tumor size was calculated using theformula:

${Tumor}\mspace{14mu} {{Volume}( {mm}^{3} )}{= \frac{w^{2} \times l}{2}}$

where w=width and l=length, in mm, of a tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Test Articles

The integrin-binding polypeptide-Fc fusion NOD201M (code-named KW2, Lot.No. BP-046-016-2), Proleukin (Lot. No. 502519AA) and anti-PD-1 (Lot. No.614616J2). NOD201M, Proleukin and anti-PD-1 were protected from lightand stored at 4° C. All agents were prepared according to protocolinstructions.

On each day of dosing, NOD201M was diluted in phosphate buffered saline(PBS) to yield a 2.08 mg/mL dosing solution. Dosing solutions werestored at 4° C.

On each day of dosing, Proleukin was dissolved in sterile water to yielda 0.04 mg/mL dosing solution. Dosing solutions were stored at 4° C.

On each day of dosing, anti-PD-1 was diluted in PBS to yield a 2 mg/mLdosing solution. Dosing solutions were stored at 4° C.

Treatment

Eleven groups of C57BL/6 mice (n=10) were dosed beginning on D1according to the MC38-NODU-e202 protocol in FIG. 13. Vehicle (PBS) andNOD201M (dosed at 500 or 1000 μg/animal), were administeredintravenously (i.v.) in a dosing volume of 0.24 mL/mouse. Proleukin(dosed at 4 or 40 μg/animal), was administered i.v. or subcutaneously(s.c.) in a dosing volume of 0.1 mL/mouse. Anti-PD-1 (dosed at 200μg/animal), was administered i.v. in a dosing volume of 0.1 mL/mouse.All volumes were dosed not adjusted according to the body weights of theindividual animals.

Group 1 animals served as controls and received vehicle, i.v on Days 1,7, 13, 19.

Group 2 animals received NOD201M at 500 μg/animal i.v. on Days 1, 7, 13,19.

Group 3 animals received Proleukin at 40 μg/animal i.v. on Days 2-4,8-10, 14-16, 20-22.

Group 4 animals received Proleukin at 4 μg/animal s.c. on Days 2-4,8-10, 14-16, 20-22.

Group 5 animals received NOD201M at 500 μg/animal i.v. on Days 1, 7, 13,19 in combination with Proleukin at 40 μg/animal i.v. on Days 2-4, 8-10,14-16, 20-22.

Group 6 animals received NOD201M at 500 μg/animal i.v. on Days 1, 7, 13,19 in combination with Proleukin at 4 μg/animal s.c. on Days 2-4, 8-10,14-16, 20-22.

Group 7 animals received anti-PD-1 at 200 μg/animal i.v. on Days 1, 7,13, 19.

Group 8 animals received NOD201M at 500 μg/animal i.v. on Days 1, 7, 13,19 in combination with anti-PD-1 at 200 μg/animal i.v. on Days 1, 7, 13,19.

Group 9 animals received NOD201M at 500 μg/animal i.v. on Days 1, 7, 13,19 in combination with anti-PD-1 at 200 μg/animal i.v. on Days 1, 7, 13,19 and Proleukin at 4 μg/animal s.c. on Days 2-4, 8-10, 14-16, 20-22.

Group 10 animals received NOD201M at 1000 μg/animal i.v. on Days 1, 7,13, 19 in combination with anti-PD-1 at 200 μg/animal i.v. on Days 1, 7,13, 19 and Proleukin at 4 μg/animal s.c. on Days 2-4, 8-10, 14-16,20-22.

Group 11 animals received NOD201M at 1000 μg/animal i.v. on Days 1, 7,13, 19 in combination with Proleukin at 4 μg/animal s.c. on Days 2-4,8-10, 14-16, 20-22.

Endpoint and Tumor Growth Delay (TGD) Analysis

Tumors were measured using calipers twice per week, and each animal waseuthanized when its tumor reached the endpoint volume of 1000 mm³ or atthe end of the study (Day 85), whichever came first. Animals that exitedthe study for tumor volume endpoint were documented as euthanized fortumor progression (TP), with the date of euthanasia. The time toendpoint (TTE) for analysis was calculated for each mouse by thefollowing equation:

${TTE} = \frac{{\log_{10}( {{end}\mspace{14mu} {point}\mspace{14mu} {volume}} )} - b}{m}$

where TTE is expressed in Days, endpoint volume is expressed in mm³, bis the intercept, and m is the slope of the line obtained by linearregression of a log-transformed tumor growth data set.

The data set consisted of the first observation that exceeded theendpoint volume used in analysis and the three consecutive observationsthat immediately preceded the attainment of this endpoint volume. Thecalculated TTE is usually less than the TP date, the day on which theanimal was euthanized for tumor size. Animals with tumors that did notreach the endpoint volume were assigned a TTE value equal to the lastday of the study (Day 85). In instances in which the log-transformedcalculated TTE preceded the day prior to reaching endpoint or exceededthe day of reaching tumor volume endpoint, a linear interpolation wasperformed to approximate the TTE. Any animal classified as having diedfrom NTR (non-treatment-related) causes due to accident (NTRa) or due tounknown etiology (NTRu) were excluded from TTE calculations (and allfurther analyses). Animals classified as TR (treatment-related) deathsor NTRm (non-treatment-related death due to metastasis) were assigned aTTE value equal to the day of death.

Treatment outcome was evaluated from tumor growth delay (TGD), which isdefined as the increase in the median time to endpoint (TTE) in atreatment group compared to the control group:

TGD=T−C,

expressed in Days, or as a percentage of the median TTE of the controlgroup:

${\% \mspace{11mu} {TGD}} = {\frac{T - C}{C} \times 100}$

where:

-   -   T=median TTE for a treatment group, and    -   C=median TTE for the designated control group.

MTV and Criteria for Regression Responses

Treatment efficacy may be determined from the tumor volumes of animalsremaining in the study on the last day. The MTV (n) was defined as themedian tumor volume on the last day of the study in the number ofanimals remaining (n) whose tumors had not attained the endpoint volume.

Treatment efficacy may also be determined from the incidence andmagnitude of regression responses observed during the study. Treatmentmay cause partial regression (PR) or complete regression (CR) of thetumor in an animal. In a PR response, the tumor volume was 50% or lessof its Day 1 volume for three consecutive measurements during the courseof the study, and equal to or greater than 13.5 mm³ for one or more ofthese three measurements. In a CR response, the tumor volume was lessthan 13.5 mm³ for three consecutive measurements during the course ofthe study. An animal with a CR response at the termination of a studywas additionally classified as a tumor-free survivor (TFS). Animals weremonitored for regression responses.

Toxicity

Animals were weighed daily for the first five Days of the study andtwice weekly thereafter. The mice were observed frequently for healthand overt signs of any adverse treatment related (TR) side effects, andnoteworthy clinical observations were recorded. Individual body weightloss was monitored per protocol, and any animal with weight lossexceeding 30% for one measurement, or exceeding 25% for threemeasurements, was to be euthanized for health as a TR death. If groupmean body weight recovered, dosing may resume in that group, but at alower dose or less frequent dosing schedule. Acceptable toxicity wasdefined as a group mean BW loss of less than 20% during the study andnot more than one TR death among ten treated animals, or 10%. Any dosingregimen resulting in greater toxicity is considered above the maximumtolerated dose (MTD). A death was to be classified as TR if it wasattributable to treatment side effects as evidenced by clinical signsand/or necropsy, or may also be classified as TR if due to unknowncauses during the dosing period or within 14 Days of the last dose. Adeath was classified as NTR if there was evidence that the death wasrelated to the tumor model, rather than treatment-related. NTR deathsare further categorized as NTRa (due to accident or human error), NTRm(due to necropsy-confirmed tumor dissemination by invasion ormetastasis), and NTRu (due to unknown causes).

It was found that not minimum toxic dosage was reached up to 100 mg/kg,with non-significant or minimal effects on complete blood count (CBC)and chemistry panel.

NOD201 is highly stable to serum and thermal challenge (stability drivenby Fc domain and not disulfide-bonded peptide). No aggregation ordegradation of NOD201 seen following extended incubation at 40° C. or 5×freeze-thaw cycles.

In silico immunogenicity analyses of NOD201 peptide (Antitope): “iTope™and TCED™ analyses were applied to the sequence in order to identifypeptides that were predicted to bind to human MHC class II and/or sharehomology to known T cell epitopes. In this analysis, no matches to knownT cell epitopes in the TCED™ were identified.” There were nonon-germline promiscuous MHC Class II binding peptides identified. Therisk of immunogenicity for NOD201 is therefore low.

Statistical and Graphical Analyses

Prism (GraphPad) for Windows 7.01 was used for graphical presentationsand statistical analyses. Survival was analyzed by the Kaplan-Meiermethod. The logrank (Mantel-Cox) and Gehan-Breslow-Wilcoxon testsdetermined the significance of the difference between the overallsurvival experiences (survival curves) of two groups, based on TTEvalues. All test results are shown in Appendix B. Two-tailed statisticalanalyses were conducted at significance level P=0.05. The analyses werenot corrected for multiple comparisons. Prism summarizes test results asnot significant (ns) at P>0.05, significant (symbolized by “*”) at0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01, and extremelysignificant (“***”) at P≤0.001. Because tests of statisticalsignificance do not provide an estimate of the magnitude of thedifference between groups, all levels of significance were described aseither significant or not significant within the text of this report.Groups with regimens that exceeded the limits for acceptable toxicitywere not evaluated statistically.

A scatter plot was constructed to show TTE values for individual mice,by group (FIG. 1). Group median tumor volumes were plotted as functionsof time (FIG. 2, upper panel). When an animal exited the study becauseof tumor size, the final tumor volume recorded for the animal wasincluded with the data used to calculate the median volume at subsequenttime points. A Kaplan-Meier plot was constructed to show the percentageof animals in each group remaining on study versus time (FIG. 17, lowerpanel).

Group median tumor volumes were plotted as a function of time, and weretruncated after the second TR death in a group. Mean plots were alsoincluded for this study (FIG. 3). Group mean BW changes over the courseof the study were graphed as percent change, ±SEM, from Day 1 (FIG. 19).Tumor growth and BW change curves excluded data for animals assessed asNTR deaths, and were truncated after more than half the mice in a groupexited the study.

Example 3 B16F10 Experimental Description

Female C57BL/6 mice (C57BL/6/NCrl, Charles River) were 8-12 weeks old atthe start of the study. Study start is the day of tumor cell implant(Day 1). Animals (n=10 per group) were randomized into treatment groupsbase on Day 1 bodyweight. B16F10 melanoma cells used for implantationwere provided by Charles River Laboratories and were harvested duringlog phase growth and resuspended in media. Mice were anesthetized withisoflurane prior to implantation.

Each mouse was injected subcutaneously in the right flank with 1×10⁶tumor cells (0.1 mL cell suspension) with 0% Matrigel. Treatment startedfour days after implantation. Animal body weight was measured at leasttwice per week during the study. Tumors were measured with calipers atleast twice weekly in two dimensions. Tumor size was calculated usingthe formula:

${Tumor}\mspace{14mu} {{Volume}( {mm}^{3} )}{= \frac{w^{2} \times l}{2}}$

where w=width and l=length, in mm, of a tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Group 1 animals served as controls and received vehicle (phosphatebuffered saline), i.v on Days 4, 10, 16, 22.

Group 2 animals received NOD201M at 1000 μg/animal i.v. on Days 4, 10,16, 22.

Group 3 animals received anti-PD-1 antibody at 200 μg/animal i.v. onDays 4, 10, 16, 22. Anti-PD-1 was clone RMP1-14 (rat IgG)—BioXcell cat#BP0146

Group 4 animals received Proleukin at 4 μg/animal s.c. on Days 5-7,11-13, 17-19, 23-25.

Group 5 animals received NOD201M at 1000 μg/animal i.v. on Days 4, 10,16, 22 in combination with anti-PD-1 antibody at 200 μg/animal i.v. onDays 4, 10, 16, 22.

Group 6 animals received NOD201M at 1000 μg/animal i.v. on Days 4, 10,16, 22 in combination with Proleukin at 4 μg/animal s.c. on Days 5-7,11-13, 17-19, 23-25.

Group 7 animals received NOD201M at 500 μg/animal i.v. on Days 4, 10,16, 22 in combination with anti-PD-1 antibody at 200 μg/animal i.v. onDays 4, 10, 16, 22, and Proleukin at 4 μg/animal s.c. on Days 5-7,11-13, 17-19, 23-25.

Measurement of Tumor Cell Infiltrates Following Treatment

Animals received NOD201M at 1000 μg/animal i.v. on Days 1, 7 alone or incombination with anti-PD-1 at 200 μg/animal i.v. on Days 1, 7, and/orProleukin at 4 μg/animal s.c. on Days 2-4, 8-9. On day 9, 24 hoursfollowing the Proleukin injection, tumors were harvested, preserved, andprocessed to single cell suspensions. Cell suspensions were stained forcell surface markers using antibodies described below, and analyzed byflow cytometry. Data is represented as the % of CD45+ cells in thetumor.

TABLE 6 Panel: CD4, CD8, Treg, total MDSC, and NK Cell populationPhenotypic Markers Antibody panel CD4 CD45⁺CD3⁺CD4⁺CD8⁻ CD45, CD3, CD4,CD8, CD11b, CD25, CD8 CD45⁺CD3⁺CD4⁻CD8⁺ Gr-1, FoxP3, CD49b(DX5), LIVE/Tregs CD45⁺CD3⁺CD4⁺CD25⁺FoxP3⁺ DEAD MDSC CD45⁺CD3-CD11b⁺Gr-1⁺ NKCD49b(DX5)+ and CD11b^(low) FoxP3, internal marker

Example 4

Purpose: Determine the efficacy of NOD201M alone and in combination withanti-PD-1, anti-PD-L1, anti-CTLA4, anti-LAG3, anti-TIM3, anti-TIGIT, andanti-CD137 in the MC38 syngeneic colon model using female C57BL/6 mice.Data shown in FIGS. 23-24.

TABLE 7 Drugs and Treatment Regimen 1 Regimen 2 Gr. N Agent μg/animalRoute Schedule Agent μg/animal Route Schedule 1^(#) 12 PBS — iv days 1,7, 13, 19 — — — — 2 12 NOD201M 500 iv days 1, 7, 13, 19 — — — — 3 12NOD201M 500 iv days 1, 7, 13, 19 anti-PD-1 200 iv days 1, 7, 13, 19 4 12NOD201M 500 iv days 1, 7, 13, 19 anti-PD-L1 200 iv days 1, 7, 13, 19 512 NOD201M 500 iv days 1, 7, 13, 19 anti-CTLA-4 200 iv days 1, 7, 13, 196 12 NOD201M 500 iv days 1, 7, 13, 19 anti-LAG-3 200 iv days 1, 7, 13,19 7 12 NOD201M 500 iv days 1, 7, 13, 19 anti-TIM-3 200 iv days 1, 7,13, 19 8 12 NOD201M 500 iv days 1, 7, 13, 19 anti-TIGIT 500 iv days 1,7, 13, 19 9 12 NOD201M 500 iv days 1, 7, 13, 19 anti-CD137 250 iv Days3, 9, 15, 21 ^(#)Control group

Procedures:

-   -   Anesthetized mice with isoflurane for implant of cells to reduce        the ulcerations.    -   Set up 240 CR female C57BL/6 mice with 1×106 MC38 tumor cells in        0% Matrigel sc in flank.    -   Cell Injection Volume was 0.1 mL/mouse.    -   Age at Start Date: 8 to 12 weeks.    -   Perform a pair match when tumors reached an average size of        60-180 mm³, and begin treatment.        -   Target ˜100 mm³ (˜6-7 days post cell implant).    -   Body Weight: 5/2 then tiwk to end.    -   Caliper Measurement: tiwk to end.    -   Any individual animal with a single observation of >than 30%        body weight loss or three consecutive measurements of >25% body        weight loss was be euthanized.    -   Any group with a mean body weight loss of >20% or >10% mortality        will stop dosing.        -   The group is not euthanized and recovery is allowed. Within            a group with >20% weight loss, individuals hitting the            individual body weight loss endpoint will be euthanized.        -   If the group treatment related body weight loss is recovered            to within 10% of the original weights, dosing may resume at            a lower dose or less frequent dosing schedule.        -   Exceptions to non-treatment body weight % recovery may be            allowed on a case-by-case basis.    -   Endpoint TGD. Animals were monitored individually. The endpoint        of the experiment was a tumor volume of 1000 mm3 or 55 days,        whichever came first. Responders can be followed longer. When        the endpoint was reached, the animals were to be euthanized per        SOP.

Mouse Dosing Instructions:

-   -   Prepared dosing solutions:        -   anti-CTLA-4, anti-LAG-3, anti-PD-1, anti-PD-L1, anti-TIGIT,            anti-TIM-3, anti-CD137, and NOD201M (store at 4° C., protect            from light)    -   vehicle=PBS    -   Dosing volume for all antibodies=0.1 mL/mouse. Do not adjust for        body weight.    -   Dosing volume KW2=0.25 mL/mouse. Do not adjust for body weight.    -   Dose regimen 2 first followed by regimen 1.

Antibodies: All from BioXcell. In Vivo MAb anti-mouse LAG-3 (SKU:BE0174-R025 mg). InVivoMAb anti-mouse TIM-3 (CD366) (SKU: BE0115-R025mg). InVivoMAb anti-mouse TIGIT (SKU: BE0274-R050 mg). InVivoMAbanti-mouse CTLA-4 (CD152) (SKU: BE0131-R025 mg). InVivoMAb anti-mousePD-L1 (B7-H1) (SKU: BE0101-R025 mg). InVivoMAb anti-mouse 4-1BB (CD137)(SKU: BE0169-R050 mg).

Example 5 MC38-NODU-010

Purpose: Collect samples for flow cytometry from female C57BL/6 micetreated with NOD201M alone and in combination with anti-PD-1 and bearingMC38 syngeneic colon tumors. Data shown in FIG. 25.

TABLE 8 Drugs and Treatment Regimen 1 Regimen 2 Gr. N Agent μg/animalRoute Schedule Agent μg/animal Route Schedule 1^(#) 10 vehicle — iv days1, 7 — — — — 2 10 anti-PD-1 200 iv days 1, 7 — — — — 3 10 NOD201M 500 ivdays 1, 7 — — — — 4 10 NOD201M 500 iv days 1, 7 anti-PD-1 200 iv days 1,7 ^(#)Control group

Procedures:

-   -   Anesthetized mice with isoflurane for implant of cells to reduce        the ulcerations.    -   Set up CR female C57BL/6 mice with 1×106 MC38-NODU tumor cells        in 0% Matrigel sc in flank.        -   Schedule cell implant so that day 9 sampling occurred early            in the week.    -   Cell Injection Volume is 0.1 mL/mouse.    -   Age at Start Date: 8 to 12 weeks.    -   Performed a pair match when tumors reach an average size of        60-180 mm³, and begin treatment.        -   Target ˜100 mm³ (˜6-7 days post cell implant).    -   Body Weight: qd to end.    -   Caliper Measurement: days 1, 3, 5, 7, 9.    -   Any individual animal with a single observation of >than 30%        body weight loss or three consecutive measurements of >25% body        weight loss was euthanized.    -   Any group with a mean body weight loss of >20% or >10% mortality        would stop dosing. The group was not euthanized and recovery was        allowed.        -   Within a group with >20% weight loss, individuals hitting            the individual body weight loss endpoint will be euthanized.            If the group treatment related body weight loss is recovered            to within 10% of the original weights, dosing could resume            at a lower dose or less frequent dosing schedule.        -   Exceptions to non-treatment body weight % recovery could be            allowed on a case-by-case basis.    -   Endpoint TGI. Animals are to be monitored as a group.        -   The endpoint of the experiment was a mean tumor weight in            Control Group of 1000 mm3 or 9 days, whichever comes first.        -   When the endpoint was reached, all the animals were to be            euthanized.

Dosing Instructions:

-   -   Compounds in Salt form: None    -   Prepare dosing solutions:        -   anti-PD-1-NODU, KW2, Proleukin-NODU—store at 4° C., protect            from light        -   anti-PD-1-NODU=anti-PD-1-NODU in PBS        -   NOD201M in PBS (internally referred to as KW2)        -   Do not freeze. Provided pre-formulated, ready to use.    -   vehicle=PBS    -   Dosing volume=0.1 mL/mouse. Do not adjust for body weight.

Sampling Instructions:

-   -   Sampling 1        -   Timepoint: day 9        -   Animals:            -   Group 1-4: 10 animals/group        -   Organ Collection            -   Tumor (weigh samples—mg): process to single cell                suspensions, shipping condition—room temp. Send to                CRL-NC for flow cytometry. Schedule with In Vitro lab.                See panel below.

TABLE 9 Panel 1: CD4, CD8, Treg, and NK Cell population PhenotypicMarkers Antibody panel CD4⁺ T cells CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻ CD45, CD11b,CD3, CD4, CD8, CD8⁺ T cells CD45⁺CD11b⁻CD3⁺CD4⁻CD8⁺ CD25, FoxP3*, CD49b,CD335, T_(reg) CD45⁺CD11b⁻CD3⁺CD4⁺CD25⁺FoxP3⁺ Live/Dead NKCD45⁺CD3⁻CD49b⁺CD335⁺ *FoxP3, internal marker

TABLE 10 Panel 2: M1 and M2 Macrophage, DC, gMDSC, mMDSC Cell populationPhenotypic Markers Antibody panel M1 MacrophageCD45⁺F4/80⁺Gr1⁻CD11b⁺CD206⁻ CD45, CD3, CD11b, F4/80, CD206*, M2Macrophage CD45⁺F4/80⁺Gr1⁻CD11b⁺CD206⁺ CD11c, I-A/I-E, Ly6C, Ly6G, DCCD45⁺CD3⁻CD11c⁺I-A/I-E⁺ Live/Dead gMDSCCD45⁺CD3⁻CD11b⁺F4/80⁻Ly6C^(low)Ly6G⁺ mMDSCCD45⁺CD3⁻CD11b⁺F4/80⁻Ly6C⁺Ly6G⁻ *CD206 internal marker

Flow data was analyzed two ways: 1) % CD45 cells and 2) cell #/gramtumor. Total MDSC and total macrophage populations in addition to thesubsets were reported. The gMDSC population can also be/contain theneutrophil population.

Example 6 Combination Therapies

Combination therapy with NOD201 and IFNα, anti-TIM3, anti-LAG3,anti-TIGIT, anti-PD1, anti-PDL1, and anti-CTLA4. See data in FIGS. 24and 40. Note: NOD201M refers to the murine Fc domain needed for thesyngeneic models.

Female C57BL/6 mice (C57BL/6/NCrl, Charles River) were eight weeks oldon Day 1 of the study and had a body weight (BW) range of 14.5-21.4 g.The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) andNIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crudeprotein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed onirradiated Enrich-o′cobs™ bedding in static microisolators on a 12-hourlight cycle at 20° C.-22° C. (68° F.-72° F.) and 40%-60% humidity.

MC38 murine colon carcinoma cells were grown in Dulbecco's ModifiedEagle Medium (DMEM) supplemented with 10% fetal bovine serum and 2 mMglutamine, 100 units/mL penicillin G sodium, 100 μg/mL streptomycinsulfate, and 25 μg/mL gentamicin. Cell cultures were maintained intissue culture flasks in a humidified incubator at 37° C., in anatmosphere of 5% CO₂ and 95% air.

The MC38 colon cells used for implantation were harvested during logphase growth and resuspended in cold RPMI media. Mice were anesthetizedwith isoflurane prior to implantation. Each mouse was injectedsubcutaneously in the right flank with 1×10⁶ tumor cells (0.1 mL cellsuspension) and tumors were monitored as their volumes approached thetarget range of 60 mm³ to 180 mm³. Six days after tumor implantation, onDay 1 of the study, animals with individual tumor volumes ranging from75 mm³ to 144 mm³ were sorted into ten groups (n=12) with group meantumor volumes ranging from 97 mm³-103 mm³.

Tumors were measured with calipers twice weekly in two dimensions. Tumorsize was calculated using the formula:

Tumor Volume (mm³)=(w ² ×l)/2

-   -   where w=width and 1=length, in mm, of a tumor.

Tumor weight may be estimated with the assumption that 1 mg isequivalent to 1 mm³ of tumor volume.

Test articles: NOD201M (Lot. No. BP-046-016-5a) and IFN-α (produced inE. coil). All antibodies were from BioXCell: anti-CD137 (LOB12.3, Cat#BE0169; Lot. No. 598916D1), anti-CTLA-4 (Clone 9H10; Cat #BE0131; Lot.No. 624316D1B), anti-LAG-3 (Clone C9B7W; Cat #BE0174; Lot. No.635116D1), anti-PD-1 (Clone RMP1-14; Cat #BE0146; Lot. No. 61461601),anti-PD-L1 (Clone 10F.9G2; Cat #BE0101; Lot. No. 615416D1), anti-TIGIT(Clone 1G9; Cat #BE0274; Lot. No. 640017J1), anti-TIM-3 (Clone RMT3-23;Cat #BE0115; Lot. No. 595616A2), All agents except IFN-α were protectedfrom light and stored at 4° C. Agent IFN-α was protected from light andstored at −20° C. All agents were prepared according to protocolinstructions.

Dosing:

On Day 1 of the study, C57BL/6 mice bearing established MC38 tumors weresorted into ten groups, n=12/group. All agents were administeredintravenously (i.v.). All antibodies and IFN-α therapy were administeredfirst, followed by NOD201M treatment.

-   -   Group 1 mice served as controls and received vehicle (PBS),        i.v., on Days 1, 7, and 13.    -   Group 2 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        13, and 19.    -   Group 3 received NOD201M at the doses and schedules as Group 2,        in combination with anti-PD-1 at 200 μg/animal, i.v., on Days 1,        7, 13, and 19.    -   Group 4 received NOD201M at 500 μg/animal, i.v., on Days 1, and        7, in combination with anti-PD-L1 at 200 μg/animal, i.v., on        Days 1, 7, and 13.    -   Group 5 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        and 13, in combination with anti-CTLA-4 at 200 μg/animal, i.v.,        on Days 1, 7, and 13.    -   Group 6 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        13, and 19, in combination with anti-LAG-3 at 200 μg/animal,        i.v., on Days 1, 7, 13, and 19.    -   Group 7 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        13, and 19, in combination with anti-TIM-3 at 200 μg/animal,        i.v., on Days 1, 7, 13, and 19.    -   Group 8 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        13, and 19, in combination with anti-TIGIT at 500 μg/animal,        i.v., on Days 1, 7, 13, and 19.    -   Group 9 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        and 13, in combination with    -   IFN-α at 50 μg/animal, i.v., on Days 3, and 9.    -   Group 10 received NOD201M at 500 μg/animal, i.v., on Days 1, 7,        and 13, in combination with anti-CD137 at 250 μg/animal, i.v.,        on Days 3, and 9.

Analysis:

Tumors were measured using calipers twice per week, and each animal waseuthanized when its tumor reached the endpoint volume of 1000 mm3 or atthe end of the study (Day 54), whichever came first. Animals that exitedthe study for tumor volume endpoint were documented as euthanized fortumor progression (TP), with the date of euthanasia.

Data:

When an animal exited the study because of tumor size, the final tumorvolume recorded for the animal was included with the data used tocalculate the median volume at subsequent time points. A Kaplan-Meierplot was constructed to show the percentage of animals in each groupremaining on study versus time (see, FIGS. 24 and 40). Group mediantumor volumes were plotted as a function of time, and were truncatedafter the second TR death in a group. Mean tumor volume plots were alsoincluded for this study (Figure provided).

These experiments demonstrate that NOD201M effectively combines withαPD1 (antt-PD1 antibody), aPDL1 (anti-PD-L1 antibody), aCTLA4(anti-CTLA4 antibody), aCD137, and IFN-α. These results are alsodemonstrated in FIG. 27, with corresponding monotherapy controls showinglack of efficacy (with the exception of IFNα, which was not repeated inFIG. 27). NOD201M does not combine with the anti-TIM3, anti-TIGIT, andanti-LAG3 antibodies in the model tested.

Example 7 Experimental Design for Generation of RNA SEQ and TIL Data

Female C57BL/6 mice (C57BL/6/NCrl, Charles River) were nine weeks old onDay 1 of the study and had a body weight (BW) range of 16.4-23.8 g. Theanimals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31Modified and Irradiated Lab Diet® consisting of 18.0% crude protein,5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiatedEnrich-o′cobs™ bedding in static microisolators on a 12-hour light cycleat 20° C.-22° C. (68° F.-72° F.) and 40-60% humidity.

MC38 murine colon carcinoma cells were grown in Dulbecco's ModifiedEagle Medium (DMEM) supplemented with 10% fetal bovine serum and 2 mMglutamine, 100 units/mL penicillin G sodium, 100 μg/mL streptomycinsulfate, and 25 μg/mL gentamicin. Cell cultures were maintained intissue culture flasks in a humidified incubator at 37° C., in anatmosphere of 5% CO₂ and 95% air.

The MC38 colon cells used for implantation were harvested during logphase growth and resuspended in cold RPMI media. Mice were anesthetizedwith isoflurane prior to cell implantation. Each mouse was injectedsubcutaneously in the right flank with 1×106 tumor cells (0.1 mL cellsuspension) and tumors were monitored as their volumes approached thetarget range of 60 to 180 mm3. Seven days after tumor implantation, onDay 1 of the study, animals with individual tumor volumes ranging from75 to 172 mm3 were sorted into four groups (n=30) with group mean tumorvolumes ranging from 120-122 mm3.

Tumors were measured with calipers twice weekly in two dimensions. Tumorsize was calculated using the formula:

Tumor Volume (mm3)=(w2×l)/2

-   -   where w=width and 1=length, in mm, of a tumor. Tumor weight may        be estimated with the assumption that 1 mg is equivalent to 1        mm3 of tumor volume.

NOD201M (Lot. No. BP-046-016-5b), and anti-PD-1 clone RMP1-14 (BioXcellLot. No. 61461601).

Treatment: Four groups (n=30) of C57BL/6 mice bearing MC38 tumors weredosed beginning on Day 1 according to the MC38-NODU-e208 protocol inFIG. 13. Vehicle (PBS) and NOD201M (dosed at 500 μg/animal), wereadministered intravenously (i.v.) at 250 μL/animal dosing volumes.Anti-PD-1 (dosed at 200 μg/animal), was also administered i.v., in adosing volume of 100 μL/mouse. All doses were provided on Days 1 and 7.Group 1 animals served as controls and received vehicle. Group 2 animalsreceived anti-PD-1 at 200 μg/animal. Group 3 received NOD201M at 500μg/animal. Group 4 animals received NOD201M at 500 μg/animal incombination with anti-PD-1 at 200 μg/animal.

Sampling: On Day 9, all animals from Groups 1-4 were euthanized andtumors were immediately removed aseptically and weighed. Tentumors/group were preserved in RNA-later and shipped at 4° C. toGenewiz. Another ten tumor samples were processed to single cellsuspensions for flow cytometry analysis at CR Discovery Services

Sample prep for flow cytometry: Mouse tumor samples were dissociatedaccording to the manufacturer's instructions using the gentleMACS™protocol “Tumor Dissociation Kit”. Briefly, tumors were excised and cutinto small pieces (2-4 mm). Tumor samples were placed into an enzymaticbuffer and processed on the gentleMACS Dissociator. Samples wereincubated for 20 minutes at 37° C. with continuous rotation.

Samples were washed twice in PBS to remove enzyme buffer, and the finalsingle cell suspensions were prepared at ˜2×107 cells/mL in StainingBuffer (2.5% FBS, 0.09% NaN3, in PBS pH 7.4). Cells were then stainedfor Live/Dead analysis and Fc receptors were blocked using TruStain Fc.Cells were the stained with the desired antibodies against cell surfacemarkers. Isotype-control antibodies were used as negative stainingcontrols when deemed necessary. All data were collected on a FACSCantoII (BD) and analyzed with FlowJo software (Tree Star, Inc.). Data wasreported as % CD45+ cells and cell # per gram of tumor. Total MDSCs andtotal macrophage populations were reported in addition to the subsets.Note that the gMDSC population can also be/contain the neutrophilpopulation.

TABLE 11 Panel 1: CD4, CD8, Treg, and NK Cell population PhenotypicMarkers Antibody panel CD4⁺ T cells CD45⁺CD11b⁻CD3⁺CD4⁺CD8⁻ CD45, CD11b,CD3, CD4, CD8, CD8⁺ T cells CD45⁺CD11b⁻CD3⁺CD4⁻CD8⁺ CD25, FoxP3*, CD49b,CD335, T_(reg) CD45⁺CD11b⁻CD3⁺CD4⁺CD25⁺FoxP3⁺ Live/Dead NKCD45⁺CD3⁻CD49b⁺CD335⁺ *FoxP3, internal marker

TABLE 12 Panel 2: M1 and M2 Macrophage, DC, gMDSC, mMDSC Cell populationPhenotypic Markers Antibody panel M1 MacrophageCD45⁺F4/80⁺Gr1⁻CD11b⁺CD206⁻ CD45, CD3, CD11b, F4/80, M2 MacrophageCD45⁺F4/80⁺ Gr1⁻CD11b⁺CD206⁺ CD206*, CD11c, I-A/I-E, Dendritic cellCD45⁺CD3⁻CD11c⁺I-A/I-E⁺ Ly6C, Ly6G, Live/Dead gMDSCCD45⁺CD3⁻CD11b⁺F4/80⁻Ly6C^(low)Ly6G⁺ mMDSCCD45⁺CD3⁻CD11b⁺F4/80⁻Ly6C⁺Ly6G⁻*CD206 internal marker

RNA Expression Analysis 1) RNA Library Preparation and HiSeq Sequencing

Total RNA was extracted using Qiagen RNeasy Mini Kit (Qiagen). RNAsamples were quantified using Qubit 2.0 Fluorometer (Life Technologies,Carlsbad, Calif., USA) and RNA integrity was checked with 2100Bioanalyzer (Agilent Technologies, Palo Alto, Calif., USA). RNA librarypreparations, sequencing reactions, and initial bioinformatics analysiswere conducted at GENEWIZ, LLC. (South Plainfield, N.J., USA).

RNA sequencing library preparation was used NEBNext Ultra RNA LibraryPrep Kit for Illumina by following manufacturer's recommendations (NEB,Ipswich, Mass., USA). Briefly, mRNA were first enriched with Oligod(T)beads. Enriched mRNAs were fragmented for 15 minutes at 94° C. Firststrand and second strand cDNA were subsequently synthesized. cDNAfragments were end repaired and adenylated at 3′ends, and universaladapter was ligated to cDNA fragments, followed by index addition andlibrary enrichment with limited cycle PCR. Sequencing libraries werevalidated using a DNA Chip on the Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, Calif., USA), and quantified by using Qubit 2.0Fluorometer (Invitrogen, Carlsbad, Calif.) as well as by quantitativePCR (Applied Biosystems, Carlsbad, Calif., USA).

The sequencing libraries were multiplexed and clustered on one lane of aflowcell. After clustering, the flowcell was loaded on the IlluminaHiSeq instrument according to manufacturer's instructions. The sampleswere sequenced using a 2×150 Paired End (PE) configuration. Imageanalysis and base calling were conducted by the HiSeq Control Software(HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq wasconverted into fastq files and de-multiplexed using Illumina's bcl2fastq2.17 software. One mis-match was allowed for index sequenceidentification.

2) Data Analysis

Before any analysis, sequence reads were trimmed to remove possibleadapter sequences at the 3′ end and nucleotides with poor quality (errorrate<0.05) using Qiagen CLC Genomics Server 9.0. Reads shorter than 30bases were removed from further analyses. Trimmed reads were aligned tothe mouse reference genome GRCm38(ftp.ensembl.org/pub/current_fasta/mus_musculus/dna). Total read countand RPKM values were calculated.

To compare gene expression between two groups of samples, Wald test wasconducted to generate P-value, FDR p-value for each gene or transcript.Significantly expressed genes or transcripts were selected if their FDRp-value<0.05 and fold-change>1.5.

For Gene Ontology Analysis, significantly expressed genes were annotatedwith Gene Ontology Biological Process categories. Then hypergeometrictest was performed for each comparison. A list of categories that had aFDR<0.05 were obtained as over-represented Biological Processes in thesignificantly expressed genes.

RNA Seq analysis (Workflow/Summary):

1. RNA-seq library preparation workflow

Whole transcriptome sequencing with polyA selection.

-   -   1. mRNA enrichment, mRNA fragmentation, and random priming    -   2. first and second strand cDNA synthesis    -   3. End repair, 5′ phosphorylation, and dA Tailing    -   4. Adaptor Ligation, PCR enrichment, and sequencing.

2. Bioinformatics analysis workflow:

-   -   1. Sequence QC, 2. trimming low quality bases, cut adaptor        sequences, 3. Map reads to the genome and splice junction, 4.        Read density on genes/exons and annotation, 5. Splice isoform        ID, 6. Calculate total hit counts and RPKM values for        transcripts/genes, 7. Comparing transcript expression, 7. GO        annotation, Uniprot annotation.

3. Gene expression analysis

-   -   3.1 Mapping sequence reads to the reference genome and        extracting gene hit counts: Sequence reads were trimmed to        remove possible adapter sequences and nucleotides with poor        quality (error rate<0.05) at the end. After trimming, sequence        reads shorter than 30 nucleotides were discarded. Remaining        sequence reads were aligned to the reference genome for mouse        (GRCm38, ftp.ensembl.org/pub/current_fasta/mus_musculus/dna).        Total gene hit counts were measured and RPKM values were        calculated.    -   3.2 Analysis of gene expression    -   After mapping and total gene hit count calculations using CLC        Genomics, the total gene hit counts were used to compare gene        differential expression.        -   3.2.1 Hierarchical clustering analysis Unsupervised            hierarchical clustering was conducted with all samples and            all genes after normalization.        -   3.2.2 Principal component analysis (PCA) analysis was            conducted with all genes to reveal the similarity among            samples.

4. Comparison of gene expression

-   -   The following comparison of gene expression values between the        groups of samples below was conducted: (note: Group 1=PBS, Group        2=anti-PD1, Group 3=NOD201,    -   Group 4=anti-PD1/NOD201 combo    -   Group 2 vs. group 1    -   Group 3 vs. group 1    -   Group 4 vs. group 1    -   Group 4 vs. group 2    -   Group 4 vs. group 3    -   Using the Wald test, p-values and fold changes were generated.        Genes with False-Discovery-Rate (FDR)<0.05 and absolute        fold-change>1.5 were called as differentially expressed genes        for each comparison.

5. Gene Ontology analysis

-   -   Gene ontology analysis (GO) was conducted on significantly        expressed genes for each comparison. A list of GO Biological        Processes with FDR<0.05 were obtained.

6. Splice variant expression analysis

-   -   For splice variants, their express levels were measured and        expression comparisons were performed just like gene expression        comparisons. A list of differentially expressed transcripts was        obtained for each comparison (FDR<0.05, fold-change>1.5).

Example 8 Antibody Fusions:

Different applications/uses of the Ab fusions: 1) The Ab fusion can beused for half-life extension of knottins through size increase orincreased FcRn recycling. 2) A knottin can be attached to an antibodythat is specific to a cancer target to create a multi-specific proteinthat binds to integrins and another target to modulate both targets orfor synergistic effects (examples would be anti-EGFR, anti-VEGF, oranti-CTLA4 and other checkpoints, etc). 3) Building on this, a knottincan be attached to an antibody to more effectively deliver the antibodyto tumors for better efficacy 4) more effective delivery might decreaseside effects of the Ab.

Fusion of the 2.5F knottin peptide to a generic antibody was carried outfor half-life extension and to create a multi-specific protein. As shownin Figure X, there are multiple attachment points for a knottin onto theheavy or light chains of an antibody. The examples provided are providedfor illustration and are not meant to be exhaustive or comprehensive.

1. Fusion of 2.5F to the N-terminus of the light chain of a murineanti-carcinoembryonic antigen (CEA) antibody (clone sm3E)

2. Fusion of 2.5F to an anti-CTLA4 antibody (clone 9D9) at differentattachment points. Fusion to the N-terminus of the heavy chain, theC-terminus of the heavy chain, or the N-terminus of the light chain.Also included is the wild-type 9D9 antibody for comparison

DNA corresponding to the knottin 2.5F was genetically fused to the heavychain or the light chain of the antibody of interest at the locationspecified. The open reading frame was cloned into a vector suitable formammalian cell expression in CHO or HEK cells. A transient expressionsystem is demonstrated, however, a stable cell line expressing theseproteins can also be generated.

For transient expression, sufficient quantities of transfection qualityDNA was prepared to perform the transfections. Robust cultures of CHO-Sor HEK293 cells were established. Pilot (milliliter) and large scaletransfections (liter scale) were performed using an appropriate hostcell line (CHO-S or HEK293) and a lipid-based transfection reagentutilizing the plasmid DNA generated. For these constructs an unmodifiedheavy or light chain was combined with a heavy or light chain that wasfused to a knottin peptide. Once cell viabilities drop below 80%, theconditioned media was harvested and clarified and the protein titerswere determined by biolayer interferometry. Single pass Protein Achromatography was used to purify the antibody fusion from thesupernatant. The purified protein was analyzed by SDS-PAGE (reducing andnon-reducing conditions) and size exclusion chromatography.

All of the antibody fusions were well expressed in HEK and CHO cells. Asshown in the figures, following Protein A purification the antibodyfusions were >98-99% monomer, and exhibited the expected molecularweight under non-reduced and reducing conditions.

4 knottin fusion proteins were produced from these constructs. Protein 1referred to as 9D9 WT and comprises umodified LC 9D9 and HC 9D9(combines constructs 1 and 2). Protein 2 referred to asNOD201G4S3-LC-HC, and comprises 2.5F-Gly4Ser3-LC 9D9 paired with HC 9D9(combines constructs 3 and 2). Protein 3, referred to asNOD201G4S3-HC-LC, and comprises 2.5F-Gly4Ser3-HC 9D9 paired withunmodified LC 9D9 (combines constructs 4 and 1). Protein 4, referred toas HC-G4S3-NOD201-LC, and comprises HC-Gly4Ser3-2.5F 9D9 paired withunmodified LC 9D9 (combines constructs 5 and 1). The 2.5F knottinpeptide was fused to different chains and termini of an anti-CTLA-4antibody, as exemplified an annotated below:

(Bold) 2.5F peptide (italics and underline): (Gly4Ser)3 linkerCTLA-4 antibody domain Construct 1. 9D9 LC (murine anti-CTLA4 lightchain) (SEQ ID NO: 155) DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECConsturct 2. 9D9 HC (murine anti-CTLA4 heavy chain) (SEQ ID NO: 156)EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVSTAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSL GConstruct 3. 2.5F-Gly4Ser3-LC 9D9 (murineanti-CTLA4 light chain with 2.5F peptide fused to N-terminus)(SEQ ID NO: 157) GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG GGGGSGGGGSGG GGSDIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECConstruct 4. 2.5F-Gly4Ser3-HC 9D9 (murineanti-CTLA4 heavy chain with 2.5F peptide fused to N-terminus)(SEQ ID NO: 158) GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG GGGGSGGGGSGG GGSEAKLQESGPVINKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVSTAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTIS RSLGConstruct 5. HC-G1y4Ser3-2.5F 9D9 (murineanti-CTLA4 heavy chain with 2.5F peptide fused to C-terminus)(SEQ ID NO: 159) EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWGQGTLITVSTAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSL G GGGGSGGGGSGGGGSGCPRPRGDNPPLTCSQDSDCLAGCVCGPN GFCG

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

1. A method for treating cancer in a subject comprising administering tothe subject an effective amount of an integrin-binding polypeptide-Fcfusion wherein said integrin-binding polypeptide-Fc fusion isadministered in a therapeutically effective amount, wherein saidintegrin-binding polypeptide comprises a sequence selected from thegroup consisting of SEQ ID NO:130 (GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG)and SEQ ID NO:131 (GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG) and wherein saidintegrin-binding polypeptide is conjugated to an Fc domain.
 2. Themethod of claim 1, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.
 3. The method of claim 2,where said Fc domain is a human Fc domain.
 4. The method of claim 1,wherein said integrin-binding polypeptide is conjugated directly to saidFc domain.
 5. (canceled)
 6. (canceled)
 7. The method of claim 1, whereinsaid method further comprises administering an immune checkpointinhibitor or an immune checkpoint.
 8. The method of claim 7, where saidimmune checkpoint inhibitor is a PD-1 inhibitor.
 9. The method of claim8, where PD-1 inhibitor is an anti-PD-1 antibody.
 10. The method ofclaim 1, wherein said integrin-binding polypeptide-Fc fusion comprisesan integrin-binding polypeptide sequence in the presence or absence of alinker, wherein said sequence is selected from the group consisting ofGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.
 11. The method of claim 1,wherein said method further comprises administering an interleukin-2(IL-2).
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. The method ofclaim 11, wherein said IL-2 is administered at a 12 MIU/m2 or lowerdaily dose.
 16. (canceled)
 17. The method of claim 1, wherein saidmethod further comprises administering either (i) IL-2 or (ii) an immunecheckpoint inhibitor or an immune checkpoint stimulator.
 18. The methodof claim 1, wherein said method further comprises administering both (i)IL-2 and (ii) an immune checkpoint inhibitor or an immune checkpointstimulator.
 19. The method of claim 17 or claim 18, wherein said immunecheckpoint inhibitor is selected from the group consisting of ananti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody.20. The method of claim 17 or claim 18, wherein said immune checkpointstimulator is selected from the group consisting an anti-4-1BB/CD137antibody, an anti-IFNα antibody, an anti-GITR antibody, an OX40antibody, an anti-CD40 antibody, an anti-ICOS antibody, and an anti-CD28antibody.
 21. The method of claim 1, wherein said integrin-bindingpolypeptide-Fc fusion binds to at least two integrins.
 22. (canceled)23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled) 27.(canceled)
 28. A polypeptide comprising an integrin-binding polypeptidesequence in the presence or absence of a linker, wherein said sequenceis selected from the group consisting ofGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.
 29. A composition comprisingan integrin-binding polypeptide sequence in the presence or absence of alinker, wherein said sequence is selected from the group consisting ofGCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO:130),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:131),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:132),GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGS (SEQ ID NO:133),GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:134), andGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCGGGGGSGGGGSGGGGS (SEQ ID NO:135),wherein said integrin-binding polypeptide sequence is directly linked toan Fc domain, wherein said Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.
 30. (canceled)
 31. A nucleicacid encoding an integrin-binding polypeptide-Fc fusion as describedherein.
 32. An expression vector comprising a nucleic acid encoding anintegrin-binding polypeptide-Fc fusion as described herein.
 33. A hostcell comprising the expression vector of claim
 31. 34. A method ofmaking an integrin-binding polypeptide-Fc fusion as described hereincomprising a) culturing the host cell of claim 33 under conditionswherein said integrin-binding polypeptide-Fc fusion is expressed; and b)recovering said integrin-binding polypeptide-Fc fusion.
 35. A method foractivating the immune system in order to treat cancer in a subjectcomprising administering to the subject an effective amount of anintegrin-binding polypeptide-Fc fusion, wherein said integrin-bindingpolypeptide-Fc fusion is administered in a therapeutically effectiveamount, wherein said integrin-binding polypeptide comprises a sequenceselected from the group consisting of SEQ ID NO:130(GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG) and SEQ ID NO:131(GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG), and wherein said integrin-bindingpolypeptide is conjugated to an Fc domain.
 36. (canceled)
 37. (canceled)38. (canceled)
 39. (canceled)
 40. (canceled)